Therapeutic use of anti-CD22 antibodies for inducing trogocytosis

ABSTRACT

Disclosed are methods and compositions of anti-B cell antibodies, preferably anti-CD22 antibodies, for diagnosis, prognosis and therapy of B-cell associated diseases, such as B-cell malignancies, autoimmune disease and immune dysfunction disease. In certain embodiments, trogocytosis induced by anti-B cell antibodies may determine antibody efficacy, disease responsiveness and prognosis of therapeutic intervention. In other embodiments, optimal dosages of therapeutic antibody may be selected by monitoring the degree of trogocytosis induced by anti-B cell antibodies. Other characteristics of anti-B-cell antibodies that may be monitored include inducing phosphorylation of CD22, CD79a and CD79b; inducing translocation of CD22, CD79a and CD79b to lipid rafts; inducing caspase-dependent apoptosis; increasing pLyn, pERKs and pJNKs; decreasing constitutively-active p38; or inducing mitochondrial membrane depolarization, generation of reactive oxygen species, upregulation of pro-apoptotic Bax and downregulation of anti-apoptotic Bcl-xl, Mcl-1 and Bcl-2.

RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.14/884,313, filed Oct. 15, 2015, which was a divisional of U.S.application Ser. No. 13/693,476 (now U.S. issued U.S. Pat. No.9,192,664), filed Dec. 4, 2012, which claimed the benefit under 35U.S.C. 119(e) of Provisional U.S. Patent Application Ser. No.61/566,828, filed Dec. 5, 2011; 61/609,075, filed Mar. 9, 2012;61/682,508, filed Aug. 13, 2012; and 61/718,226, filed Oct. 25, 2012,each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 29, 2012, isnamed IMM337US1.txt and is 52,688 bytes in size.

FIELD OF THE INVENTION

The present invention concerns compositions and methods of use ofantibodies against B-cell surface markers, in particular, CD19, CD20,and CD22. Preferably, the antibody is an anti-CD22 antibody. Morepreferably, the anti-CD22 antibody induces trogocytosis of multiplesurface markers, which include, but are not limited to, CD19, CD20,CD21, CD22 and/or CD79b on normal, lupus, and malignant B cells (donorcells) via leukocytes, including monocytes, NK cells and granulocytes(recipient cells). Most preferably, the antibody efficacy, disease cellresponsiveness and/or prognosis for disease progression are a functionof trogocytosis induced by such antibodies. The trogocytosis-inducingantibody may be used alone, or in combination with other agents, whichinclude one or more different antibodies that may or may not havetrogocytosis-inducing activity. Where a combination of two antibodies isdesirable, a bispecific antibody derived from the two antibodies ofinterest may be used in lieu of a combination of such antibodies.Bispecific antibodies are preferred to administration of combinations ofseparate antibodies, due to cost and convenience. However, wherecombinations of separate antibodies provide improved safety or efficacy,the combination may be utilized. One preferred form of the bispecificantibody is a hexavalent antibody (HexAb) that is made as aDOCK-AND-LOCK complex. Further, a bispecific antibody capable ofbridging the donor and recipient cells may not require the presence ofFc for trogocytosis. The compositions and methods are of use in therapyand/or detection, diagnosis or prognosis of various disease states,including but not limited to autoimmune diseases, immune dysfunctiondiseases and cancers.

BACKGROUND

Trogocytosis (also referred to as shaving in the literature) is aprocess by which transfer of membrane—bound proteins and membranecomponents occur between two different types of live cells associated toform an immunological synapse. As a result, the membrane-bound proteinsand membrane components are transferred from the donor cells to therecipient cells. Both unidirectional and bidirectional trogocytosisbetween the two interacting cells may occur. One prominent example oftrogocytosis is the extraction of surface antigens fromantigen-presenting cells (APCs) by T cells (Joly & Hudrisier, 2003, NatImmunol 4:85). The process involves transfer of plasma membranefragments from the APC to the lymphocyte (Joly & Hudrisier, 2003).Intercellular transfer of T cell surface molecules to APCs has also beenreported (Nolte-′t Hoen et al, 2004, Eur J Immunol 34: 3115-25; Busch etal 2008, J Immunol 181: 3965-73) via mechanisms that may includetrogocytosis, exosomes and ectodomain shedding (Busch et al 2008, ibid).Trogocytosis can also occur between natural killer (NK) cells and tumorsand can convert activated NK cells into suppressor cells, via uptake ofthe immunosuppressive HLA-G molecule, which protects the tumor cellsfrom cytolysis (Caumartin et al., 2007, EMBO J 26:423-30). CD4+ and CD8+T cells can, respectively, acquire MHC Class II and MHC Class Imolecules from APCs in an antigen-specific manner (Caumartin et al.,2007). Trogocytosis of HLA-DR, CD80 and HLA-G1 from APCs to T cells hasbeen shown to occur in humans (Caumartin et al., 2007). After acquiringHLA-DR and CD80, T cells stimulated resting T cells in anantigen-specific manner, acting as APCs themselves (Caumartin et al.,2007). More generally, trogocytosis may act to regulate immune systemresponsiveness to disease-associated antigens and can either stimulateor suppress immune response (Ahmed et al., 2008, Cell Mol Immunol5:261-69).

The effects of trogocytosis on therapeutic antibody responsiveness andthe induction of trogocytosis by therapeutic antibodies remain poorlyunderstood. It has been suggested that induction of trogocytosis byexcess amounts of rituximab may result in removal of rituximab-CD20complexes from tumor cell surfaces by monocytes, producing antigenicmodulation (shaving) and rituximab-resistant tumor cells (Beum et al.,2006, J Immunol 176:2600-8). Thus, use of lower, more frequent doses ofrituximab to reduce antigen shaving has been suggested (Beum et al.,2006). Transfer of rituximab/CD20 complexes to monocytes is mediated byFcγR and it has also been suggested that polymorphisms in FcγRII andFcγRIII may affect the degree of antibody-induced shaving and predictresponsiveness to antibody therapy (Beum et al., 2006). In this regard,use of antibodies or other inhibitors that block trogocytosis mayenhance efficacy and reduce tumor cell escape from cytotoxicity (Beum etal., 2006). On the other hand, the functional consequences ofantibody-mediated trogocytosis to confer therapeutic benefits are lessexplored.

A need exists in the art for a better understanding of the induction oftrogocytosis by therapeutic antibodies, the effect of trogocytosis onantigen shaving, and the effects of trogocytosis and shaving ontherapeutic efficacy, target cell susceptibility, and immune systemresponses in various disease states.

SUMMARY

The present invention concerns compositions and methods of use ofantibodies against B-cell surface markers, such as CD19, CD20, CD22and/or CD79b. Preferably, the antibody is an anti-CD22 antibody. Morepreferably, the anti-CD22 antibody induces trogocytosis of multiplesurface markers, which include, but are not limited to, CD19, CD21,CD20, CD22 and CD79b on normal, lupus, and malignant B cells viamonocytes, NK cells and granulocytes. Most preferably, the anti-CD22antibody displays little or negligible direct cytotoxicity to normal Bcells based on an in vitro cell proliferation assay that shows less than20% growth inhibition when compared with untreated control, yet reducesCD19, CD21, CD20, CD22, and CD79b to 20% or more of the untreatedcontrol via trogocytosis in the presence of peripheral blood mononuclearcells (PBMCs) or purified FcγR-positive cells, such as NK cells,monocytes and granulocytes. One example of a preferred anti-CD22antibody is epratuzumab, which induces trogocytosis without incurringdirect cytotoxicity to B cells, thus providing an unexpected andsubstantial advantage in treating autoimmune diseases, such as systemiclupus erythematosus (SLE), ANCA-associated vasculitides, and otherautoimmune diseases.

In certain embodiments, administration of an antibody against aselective B cell marker, such as an anti-CD22 antibody, inducestrogocytosis in B cells, resulting in decreased levels of CD19, CD20,CD21, CD22 and CD79b on the surface of affected B cells. The reductionin these regulators of antigen-specific B-cell receptor (BCR),particularly CD19, inhibits B cell activation in response to Tcell-dependent antigens and has a therapeutic effect on autoimmune andimmune dysfunction diseases, which are mediated at least in part by Bcell activation. In certain alternative embodiments, an affibody orfynomer fused to a human Fc may be used in place of an antibody.

In a preferred embodiment, the efficacy of anti-B cell antibodies fortherapeutic use in autoimmune and/or immune dysfunction diseases ispredicted by trogocytosis-mediated decrease in the levels of BCRregulators on the cell surface, particularly that of CD19. Efficacy ofanti-B-cell antibodies, such as anti-CD22 antibodies, for therapeuticuse in specific autoimmune and/or immune dysfunction diseases may bepredicted by measuring the extent of trogocytosis of cell surface CD19in B cells. The method may involve obtaining a sample of B cells from anindividual with autoimmune or immune dysfunction disease, exposing the Bcells to an anti-B cell (particularly anti-CD22) antibody, measuring thelevels of CD19 in the B cells, and predicting the efficacy of the anti-Bcell antibody for disease therapy. Alternatively, the method may involveadministering the antibody to a subject and monitoring the level oftrogocytosis and/or antigen shaving. In other alternative embodiments,the effect of anti-B cell antibody on inducing trogocytosis of CD19 maybe used to predict the susceptibility of the diseased cell to antibodytherapy and/or the prognosis of the individual with the disease. Instill other embodiments, use of additional predictive factors such asFcγR polymorphisms may be incorporated into the method. The skilledartisan will realize that the same compositions and methods may be ofuse to provide a prognosis of autoimmune or immune dysfunction diseaseprogression and/or to select an optimum dosage of anti-B cell antibodyto administer to a patient with autoimmune and/or immune dysfunctiondiseases, including but not limited to systemic lupus erythematosus andANCA-associated vasculitides.

Exemplary autoimmune or immune dysfunction diseases include acute immunethrombocytopenia, chronic immune thrombocytopenia, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, pemphigus vulgaris, diabetes mellitus (e.g., juvenilediabetes), Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitides,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjögren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis, fibrosing alveolitis, graft-versus-hostdisease (GVHD), organ transplant rejection, sepsis, septicemia andinflammation.

In another embodiment, trogocytosis and/or antigen shaving may beutilized to select an optimal dosage of anti-B cell antibody, such asanti-CD22 antibody, to be administered to a subject with a malignancy,preferably a B-cell malignancy, such as non-Hodgkin's lymphoma, B-cellacute and chronic lymphoid leukemias, Burkitt lymphoma, Hodgkin'slymphoma, hairy cell leukemia, multiple myeloma and Waldenstrom'smacroglobulinemia. Either in vitro or in vivo analysis may be performed.For example, a sample of whole blood or PBMCs may be obtained from apatient with a B-cell malignancy and incubated with differentconcentrations of anti-B cell antibody, such as anti-CD22 antibody.Dose-response curves may be constructed based on evidence oftrogocytosis and/or antigen shaving from B cells. For example, relativecell surface expression levels of CD19, CD20, CD21, CD22 and/or CD79bmay be determined by standard assays, such as flow cytometry usingfluorescence labeled antibodies. Depending on the disease to be treated,the optimum concentration of antibody to administer to the patient maybe selected to either maximize or minimize trogocytosis and/or antigenshaving. The skilled artisan will realize that, for example, selectionof an optimal dosage of anti-CD22 antibody to administer may preferablyinvolve monitoring of relative cell surface expression of CD22.Selection of optimal dosage of anti-CD20 antibody may preferably involvemonitoring of relative cell surface expression of CD20. However, themethod is not limiting and monitoring of surrogate antigens orcombinations of antigens may provide a preferred result. For example,monitoring relative cell surface expression of CD22 may in some casespredict optimal levels of anti-CD19, anti-CD20, anti-CD21 or anti-CD79bantibody to administer, or vice-versa. The skilled artisan will realizethat the same methods and compositions may be used to determine theefficacy of an anti-B cell antibody against a B-cell malignancy, theprognosis of a B-cell malignancy, and/or the susceptibility of amalignant B cell to anti-B cell antibody.

Antibodies against B-cell surface proteins, such as CD19, CD20, CD21,CD22 and/or CD79b, are known in the art and any such known antibodymight be used in the claimed compositions and methods. An exemplaryanti-CD20 antibody is hA20 (veltuzumab), disclosed for example in U.S.Pat. No. 7,251,164, the Examples section of which is incorporated hereinby reference. Other known anti-CD20 antibodies of potential use include,but are not limited to, rituximab (Genentech, South San Francisco,Calif.), GA101 (obinutuzumab; R05072759, Roche, Basle, Switzerland),ofatumumab (GlaxoSmithKline, London, England), ocrelizumab (Roche,Nutley, N.J.), AME-133v (ocaratuzumab, MENTRIK Biotech, Dallas, Tex.),ibritumomab (Spectrum Pharmaceuticals, Irvine, Calif.) and PRO131921(Genentech, South San Francisco, Calif.). An exemplary anti-CD19antibody is hA19, disclosed for example in U.S. Pat. No. 7,109,304, theExamples section of which is incorporated herein by reference. Otherknown anti-CD19 antibodies of potential use include, but are not limitedto, XmAb5574 (Xencor, Monrovia, Calif.), 5F3 (OriGene, Rockville, Md.),4G7 (Pierce, Rockford, Ill.), 2E2 (Pierce, Rockford, Ill.), 1G9 (Pierce,Rockford, Ill.), LT19 (Santa Cruz Biotechnology, Santa Cruz, Calif.) andHD37 (Santa Cruz Biotechnology, Santa Cruz, Calif.). An exemplaryanti-CD22 antibody is hLL2 (epratuzumab), disclosed for example in U.S.Pat. No. 7,074,403, the Examples section of which is incorporated hereinby reference. Other known anti-CD22 antibodies of potential use include,but are not limited to, inotuzumab (Pfizer, Groton, Conn.), CAT-3888(Cambridge Antibody Technology Group, Cambridge, England), CAT-8015(Cambridge Antibody Technology Group, Cambridge, England), HB22.7 (DukeUniversity, Durham, N.C.) and RFB4 (e.g., Invitrogen, Grand Island,N.Y.; Santa Cruz Biotechnology, Santa Cruz, Calif.). Exemplary anti-CD21antibodies of potential use include, but are not limited to, LS-B7297(LSBio, Seattle, Wash.), HB5 (eBioscience, San Diego, Calif.), A-3(Santa Cruz Biotechnology, Santa Cruz, Calif.), D-19 (Santa CruzBiotechnology, Santa Cruz, Calif.), Bly4 (Santa Cruz Biotechnology,Santa Cruz, Calif.), 1F8 (Abcam, Cambridge, Mass.) and Bu32 (BioLegend,San Diego, Calif.). Exemplary anti-CD79b antibodies of potential useinclude, but are not limited to, B29 (LSBio, Seattle, Wash.), 3A2-2E7(LSBio, Seattle, Seattle, Wash.), CD3-1 (eBioscience, San Diego, Calif.)and SN8 (Santa Cruz Biotechnology, Santa Cruz, Calif.). Many suchantibodies are publicly known and/or commercially available and any suchknown antibody may be utilized.

An antibody of use may be chimeric, humanized or human. The use ofchimeric antibodies is preferred to the parent murine antibodies becausethey possess human antibody constant region sequences and therefore donot elicit as strong a human anti-mouse antibody (HAMA) response asmurine antibodies. The use of humanized antibodies is even morepreferred, in order to further reduce the possibility of inducing a HAMAreaction. Techniques for humanization of murine antibodies by replacingmurine framework and constant region sequences with corresponding humanantibody framework and constant region sequences are well known in theart and have been applied to numerous murine anti-cancer antibodies.Antibody humanization may also involve the substitution of one or morehuman framework amino acid residues with the corresponding residues fromthe parent murine framework region sequences. As discussed below,techniques for production of human antibodies are also well known.

The antibody may also be multivalent, or multivalent and multispecific.The antibody may include human constant regions of IgG1, IgG2, IgG3, orIgG4.

In certain embodiments, one or more anti-B-cell antibodies may beadministered to a patient as part of a combination of antibodies. Theantibodies may bind to different epitopes of the same antigen or todifferent antigens. Preferably, the antigens are selected from the groupconsisting of BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4, CD5, CD7, CD8,CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD21, CD22,CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43, CD45, CD55,CD56, CCD57, CD59, CD64, CD71, CD79a, CD79b, CD117, CD138, FMC-7 andHLA-DR. However, antibodies against other antigens of use for therapy ofcancer, autoimmune diseases or immune dysfunction diseases are known inthe art, as discussed below, and antibodies against any suchdisease-associated antigen known in the art may be utilized.

In more preferred embodiments, the allotype of the antibody may beselected to minimize host immunogenic response to the administeredantibody, as discussed in more detail below. A preferred allotype is anon-G1m1 allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. Thenon-G1m1 allotype is preferred for decreased antibody immunoreactivity.Surprisingly, repeated subcutaneous administration of concentrated nG1m1antibody was not found to induce significant immune response, despitethe enhanced immunogenicity of subcutaneous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are provided to illustrate preferred embodimentsof the invention. However, the claimed subject matter is in no waylimited by the illustrative embodiments disclosed in the drawings.

FIG. 1. Epratuzumab-induced reduction of select surface antigens onnormal B cells. Fresh PBMCs isolated from the blood of healthy donorswere treated overnight with 10 μg/mL epratuzumab or a non-bindingisotype control mAb (hMN-14) and the relative surface levels of selectedproteins on B cells were measured by flow cytometry. The effect ofepratuzumab on 13 different B cell antigens was surveyed. The number ofdonors evaluated for each specific antigen is indicated in parentheses.The % mean fluorescence intensity of the isotype control treatment isshown. Error bars, Std. Dev.

FIG. 2. Reduction of CD19 on CD27⁺ and CD27⁻ B cells from three healthydonors (N1, N2 and N3). The % mean fluorescence intensity of the isotypecontrol treatment is shown. Error bars, Std. Dev.

FIG. 3. Example of the reduction of CD19, CD22, CD21 and CD79b on CD27⁺and CD27⁻ B cells from a healthy donor. The % mean fluorescenceintensity of the isotype control treatment is shown. Error bars, Std.Dev.

FIG. 4. Comparison of the reduction of CD19 and CD21 on B cellsfollowing 2 h (N=5 donors) vs. overnight treatment (N=16 donors) with 10μg/mL epratuzumab or isotype control (hMN-14). The % mean fluorescenceintensity of the isotype control treatment is shown. Error bars, Std.Dev.

FIG. 5. Histogram showing CD22 levels on B cells gated from PBMCs ofhealthy donors following overnight treatment with 10 μg/mL epratuzumab,hA19 (anti-CD19) or isotype control (hMN-14).

FIG. 6. Fresh PBMCs isolated from healthy donors were treated overnightwith epratuzumab, veltuzumab or rituximab. The relative B cell count (Bcells) and levels of CD19, CD22, CD21 and CD79b following treatment isshown as the % mean fluorescence intensity of the isotype control(hMN-14) treatment at the same protein concentration. Error bars, Std.Dev.

FIG. 7. Fresh PBMCs isolated from healthy donors were treated overnightwith 10 μg/mL epratuzumab, 1 mg/mL epratuzumab or 10 μg/mL epratuzumabplus 1 mg/mL hMN-14. The B cell surface levels of CD19, CD21, CD22 andCD79b are shown as the % mean fluorescence intensity of the isotypecontrol (hMN-14) treatment at the same protein concentration. Errorbars, Std. Dev.

FIG. 8. PBMCs from two normal donors (N13 and N14) were treatedovernight with epratuzumab or hMN-14 at varied concentrations (1ng/mL-10 mg/mL). The B cell surface levels of CD19, CD21, CD22 and CD79bare shown as the % mean fluorescence intensity of the isotype control(hMN-14) treatment at the same protein concentration except for the 10mg/mL epratuzumab, which was derived using 1 mg/mL hMN-14 as control.Error bars, Std. Dev.

FIG. 9. PBMCs were treated with whole IgG or an F(ab′)₂ fragment ofepratuzumab at 10 μg/mL. The % mean fluorescence intensity of theisotype control (hMN-14) treatment at the same protein concentration isshown. Error bars, Std. Dev.

FIG. 10. Daudi human Burkitt lymphoma cells (1×10⁵ cells) were treatedovernight with 10 μg/mL epratuzumab or an isotype control mAb (hMN-14)in the presence, or absence, of PBMCs (1×10⁶). The plot is shown as the% mean fluorescence intensity of the isotype control treatment. Errorbars, Std. Dev.

FIG. 11. Raji human Burkitt lymphoma cells (1×10⁵ cells) were treatedovernight with 10 μg/mL epratuzumab or an isotype control mAb (hMN-14)in the presence, or absence, of PBMCs (1×10⁶) or goat-anti-human IgG (20μg/mL) as a crosslinking second antibody. The plot is shown as the %mean fluorescence intensity of the isotype control treatment. Errorbars, Std. Dev.

FIG. 12. Gating of monocytes and T cells with anti-CD3 and anti-CD14from PBMCs (top), T cell-depleted PBMCs (middle) and monocyte-depletedPBMCs (bottom).

FIG. 13. Epratuzumab-induced reduction of CD19 and CD22 with monocytes.Daudi cells (1×10⁵) were mixed with effector cells (1×10⁶) comprisingPBMCs, T cell depleted-PBMCs or monocyte-depleted PBMCs, which were eachderived from the same donor. The cell mixtures were incubated overnightwith 10 μg/mL epratuzumab or an isotype control mAb (hMN-14). The levelof CD19 and CD22 on the surface of Daudi (A) and the intrinsic B cells(B) were measured by flow cytometry and plotted as the % meanfluorescence intensity of the isotype control treatment.

FIG. 14. Purified T cells do not participate in epratuzumab-inducedtrogocytosis. Daudi cells (1×10⁵) were mixed with 1×10⁶ PBMCs orpurified T cells, or without effector cells and treated overnight with10 μg/mL epratuzumab or an isotype control mAb (hMN-14). The levels ofCD19, CD21, CD22 and CD79b on the surface of Daudi was measured by flowcytometry and plotted as the % mean fluorescence intensity of theisotype control treatment.

FIG. 15. Gating of monocytes with anti-CD3 and anti-CD14 from PBMCs(top), monocyte-depleted PBMCs (middle) and purified monocytes (bottom).

FIG. 16. Daudi cells (1×10⁵) were mixed with PBMCs (1×10⁶),monocyte-depleted PBMCs (1×10⁶) or purified monocytes (5×10⁵), whichwere each derived from the same donor. The cell mixtures were incubatedovernight with 10 μg/mL epratuzumab or an isotype control mAb (hMN-14).The level of CD19 and CD22 on the surface of Daudi were measured by flowand plotted as the % mean fluorescence intensity of the isotype controltreatment.

FIG. 17. Gating of monocytes from PBMCs. The monocyte gate (top) wasfurther separated into CD14++ and CD14+CD16+ sub-populations (bottom).

FIG. 18. (Top left) Gating by scattering from a mixture of purifiedmonocytes and Daudi. (Top right) The Daudi cells were further identifiedas CD19+CD22+ cells in the Daudi gate. (Bottom) The monocyte gate wasfurther separated into CD14++ and CD14+CD16+ sub-populations.

FIG. 19. Epratuzumab-induced trogocytosis with monocytes. Daudi cellswere mixed with purified monocytes 1:1 and treated for 1 h withepratuzumab (black dots) or hMN-14 (white dots) before analysis by flowcytometry. The monocyte gate determined by forward vs. side scatteringwas further gated with anti-CD14.

FIG. 20A. Daudi cells were mixed with purified monocytes 1:1 and treatedfor 1 h with epratuzumab (black dots) or hMN-14 (white dots) beforeanalysis by flow cytometry. The monocyte gate determined by forward vs.side scattering was further separated into CD14⁺⁺ monocyte populations,which were evaluated for CD19 and CD22 levels.

FIG. 20B. Daudi cells were mixed with purified monocytes 1:1 and treatedfor 1 h with epratuzumab (black dots) or hMN-14 (white dots) beforeanalysis by flow cytometry. The monocyte gate determined by forward vs.side scattering was further separated into CD14⁺CD16⁺ monocytepopulations, which were evaluated for CD19 and CD22 levels.

FIG. 21. The Daudi cells (CD19⁺ cells in the Daudi gate) were analyzedfor CD19 and CD22 levels following a 1-hour epratuzumab treatment withPBMCs, purified monocytes or monocyte-depleted PBMCs. The level of CD19and CD22 on the surface of Daudi were measured by flow and plotted asthe % mean fluorescence intensity of the isotype control treatment.

FIG. 22. (Top) Gating by scattering from a mixture of PBMCs and Daudi.(Bottom) The lymphocyte gate was further separated with CD14 and CD16staining to identify NK cells.

FIG. 23A. Daudi cells were mixed with PBMCs 1:5 and treated for 1 h withepratuzumab (black dots) or hMN-14 (white dots) before analysis by flowcytometry. The NK cells were identified as CD14⁻CD16⁺ cells in thelymphocyte gate, which were evaluated for the levels of CD19 and CD22.

FIG. 23B. Daudi cells were mixed with monocyte-depleted PBMCs 1:5 andtreated for 1 h with epratuzumab (black dots) or hMN-14 (white dots)before analysis by flow cytometry. The NK cells were identified asCD14⁻CD16⁺ cells in the lymphocyte gate, which were evaluated for thelevels of CD19 and CD22.

FIG. 24. Gating of granulocytes mixed with Daudi first by forward vs.side scatter (Top) followed by anti-CD16 staining.

FIG. 25A. Daudi cells were mixed with purified granulocytes 1:2 andtreated for 1 h with epratuzumab (black dots) or hMN-14 (white dots)before analysis by flow cytometry. The granulocyte gate was furtherrefined for CD16⁺ cells and evaluated for CD19 and CD22 levels.

FIG. 25B. Daudi cells were mixed with purified granulocytes 1:2 andtreated for 1 h with epratuzumab (black dots) or hMN-14 (white dots)before analysis by flow cytometry. The granulocyte gate was furtherrefined for CD16⁺ cells and evaluated for CD19 and CD79b levels.

FIG. 26. Daudi cells were mixed with purified granulocytes 1:2 andtreated for 1 h with epratuzumab or hMN-14 before analysis by flowcytometry. The Daudi cells (CD19⁺ cells in the Daudi gate) were analyzedfor CD19, CD22 and CD79b levels and graphed as the % mean fluorescenceintensity of the isotype control treatment.

FIG. 27. PBMCs were isolated from blood specimens of three naive SLEpatients and treated overnight with 10 μg/mL epratuzumab or hMN-14. Therelative levels of CD19, CD22, CD21 and CD79b on B cells post-treatmentwere measured by flow cytometry and graphed as the % mean fluorescenceintensity of the isotype control treatment.

FIG. 28. PBMCs were isolated from blood specimens of three naive SLEpatients and treated overnight with 10 μg/mL epratuzumab or hMN-14. Bcells were gated further into CD27⁺ and CD27⁻ populations beforeanalysis. The relative levels of CD19 and CD22 on the B cellsub-populations post-treatment were measured by flow cytometry andgraphed as the % mean fluorescence intensity of the isotype controltreatment.

FIG. 29. PBMCs were isolated from blood specimens of naive SLE patientsand treated overnight with 10 μg/mL epratuzumab, an F(ab′)₂ ofepratuzumab or hMN-14. B cells were gated further into CD27⁺ and CD27⁻populations before analysis. The figure shows an example from one naiveSLE patient. The relative levels of CD19, CD22, CD21 and CD79b on the Bcell sub-populations post-treatment were measured by flow cytometry andgraphed as the % mean fluorescence intensity of untreated PBMCs.

FIG. 30A. The MFI levels of CD22 were measured by flow cytometry on Bcells gated from PBMCs that were isolated from four SLE patients who hadyet to receive any treatment (naïve), five patients on activeimmunotherapy with epratuzumab and two patients on immunotherapy withBENLYSTA®. Each point represents an individual patient sample.

FIG. 30B. The MFI levels of CD19 were measured by flow cytometry on Bcells gated from PBMCs that were isolated from four SLE patients who hadyet to receive any treatment (naïve), five patients on activeimmunotherapy with epratuzumab and two patients on immunotherapy withBENLYSTA®. Each point represents an individual patient sample.

FIG. 30C. The MFI levels of CD21 were measured by flow cytometry on Bcells gated from PBMCs that were isolated from four SLE patients who hadyet to receive any treatment (naïve), five patients on activeimmunotherapy with epratuzumab and two patients on immunotherapy withBENLYSTA®. Each point represents an individual patient sample.

FIG. 30D. The MFI levels of CD79b were measured by flow cytometry on Bcells gated from PBMCs that were isolated from four SLE patients who hadyet to receive any treatment (naïve), five patients on activeimmunotherapy with epratuzumab and two patients on immunotherapy withBENLYSTA®. Each point represents an individual patient sample.

FIG. 31A. Epratuzumab (hLL2) immobilized on plastics (hLL2*) displayscytotoxicity against Daudi (D1-1) and Ramos cells. Cells (1×10⁴ cellsper well in 48-well plate) were added to the plates coated with varyingamounts of hLL2* for 4 days and viability was determined using the MTSreagent.

FIG. 31B. Epratuzumab (hLL2) immobilized on plastics (hLL2*) displayscytotoxicity against Daudi (D1-1) and Ramos cells. Effect of similarconcentrations of soluble epratuzumab on the growth of cells.

FIG. 31C. Epratuzumab (hLL2) immobilized on plastics (hLL2*) displayscytotoxicity against Daudi (D1-1) and Ramos cells. D1-1 and Ramos cells(2×10⁵ cells per well in 6-well plate) were treated with 5 and 20 μl ofhLL2* on polystyrene beads for 24 and 48 h respectively followed byAnnexin staining analysis.

FIG. 31D. Epratuzumab (hLL2) immobilized on plastics (hLL2*) displayscytotoxicity against Daudi (D1-1) and Ramos cells. D1-1 cells were addedto the plates coated with F(ab′)2 fragments of hLL2 (10 μg/mL) andapoptosis was determined 24 h later (left panel). Growth inhibition byvarying concentrations of immobilized F(ab′)2 fragments of hLL2 wasevaluated in the MTS assay (right panel). Nonspecific isotype-matchedcontrol antibody, hMN-14, was evaluated in solution as well asimmobilized to determine specificity of hLL2*. Error bars representstandard deviation (SD), where n=3.

FIG. 32A. Immobilized epratuzumab (hLL2*) induces phosphorylation ofCD79a and CD79b and their translocation into lipid rafts along withCD22. D1-1 (3×10⁷) cells were added to plates coated with hLL2* for 2 h.Co-immunoprecipitation (Co-IP) experiments were performed using p-Tyrantibody 4G10. Phosporylation was determined probing the membranes withCD22, CD79a or CD79b antibodies.

FIG. 32B. Immobilized epratuzumab (hLL2*) induces phosphorylation ofCD79a and CD79b and their translocation into lipid rafts along withCD22. Phosphorylation profile of CD22, CD79a and CD79b with solubleepratuzumab (7.5 μg/mL) along with suboptimal amounts of anti-IgM (1μg/mL) and a crosslinking secondary antibody (10 μg/mL).

FIG. 32C. Immobilized epratuzumab (hLL2*) induces phosphorylation ofCD79a and CD79b and their translocation into lipid rafts along withCD22. Cells as described earlier were treated with hLL2* and cellslysates were fractionated by sucrose density gradientultracentrifugation. The lipid rafts were collected from the interfaceof 5% to 30% sucrose. Distribution of CD22, CD79a and CD79b in the lipidrafts was analyzed by Western blotting using specific antibodies.

FIG. 32D. Immobilized epratuzumab (hLL2*) induces phosphorylation ofCD79a and CD79b and their translocation into lipid rafts along withCD22. Immunofluorescence analysis of CD22 and IgM receptors by solublehLL2 with or without anti-IgM. The receptors colocalize in the caps whenhLL2 and anti-IgM are added together.

FIG. 32E. Immobilized epratuzumab (hLL2*) induces phosphorylation ofCD79a and CD79b and their translocation into lipid rafts along withCD22. Immunofluorescence analysis of CD22 and IgM receptors by hLL2immobilized to protein A beads.

FIG. 33. Inhibition of JNK pathway inhibits hLL2* induced apoptosis.SP600125, a chemical inhibitor for stress activated JNK MAP kinase,inhibits the apoptosis in D1-1 cells induced by hLL2*. Cells werepretreated with inhibitor SP600125 (2.5 or 5 nM) for 2 h followed byaddition of cells in media containing the inhibitor to wells coated withhLL2* (10 μg/mL), and apoptosis was determined by annexin V staining 24h later. Error bars represent SD, where n=3.

FIG. 34. Immobilized epratuzumab (hLL2*) induces caspase-dependentapoptosis. Caspase inhibitor z-vad-fmk inhibits apoptosis in D1-1 cellsinduced by hLL2*. Cells were pretreated with z-vad-fmk pan-caspaseinhibitor (10 or 25 μM) for 2 h, followed by addition of cells in mediacontaining the inhibitor to wells coated with hLL2* (10 μg/mL), andapoptosis was determined by annexin V staining 24 h later. Error barsrepresent SD, where n=3.

DETAILED DESCRIPTION

Definitions

The following definitions are provided to facilitate understanding ofthe disclosure herein. Where a term is not specifically defined, it isused in accordance with its plain and ordinary meaning.

As used herein, the terms “a”, “an” and “the” may refer to either thesingular or plural, unless the context otherwise makes clear that onlythe singular is meant.

An “antibody” refers to a full-length (i.e., naturally occurring orformed by normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive (i.e., antigen-binding) portion of an immunoglobulin molecule,like an antibody fragment.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, single domain antibodies (DABs or VHHs) andthe like, including half-molecules of IgG4 (van der Neut Kolfschoten etal., 2007, Science 317:1554-1557). Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-CD22 antibody fragment binds with anepitope of CD22. The term “antibody fragment” also includes isolatedfragments consisting of the variable regions, such as the “Fv” fragmentsconsisting of the variable regions of the heavy and light chains,recombinant single chain polypeptide molecules in which light and heavychain variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a rodent antibody, are transferredfrom the heavy and light variable chains of the rodent antibody intohuman heavy and light variable domains, including human framework region(FR) sequences. The constant domains of the antibody molecule arederived from those of a human antibody.

A “human antibody” is an antibody obtained from transgenic mice thathave been genetically engineered to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. (See, e.g., McCafferty et al., Nature 348:552-553 (1990) forthe production of human antibodies and fragments thereof in vitro, fromimmunoglobulin variable domain gene repertoires from unimmunizeddonors). In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see, e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.(See, U.S. Pat. Nos. 5,567,610 and 5,229,275).

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includebut are not limited to antibodies, antibody fragments, drugs, cytokineor chemokine inhibitors, pro-apoptotic agents, tyrosine kinaseinhibitors, toxins, enzymes, nucleases, hormones, immunomodulators,antisense oligonucleotides, siRNA, RNAi, chelators, boron compounds,photoactive agents, dyes and radioisotopes.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes, contrast agents, fluorescent compoundsor molecules and enhancing agents (e.g., paramagnetic ions). Preferably,the diagnostic agents are selected from the group consisting ofradioisotopes, enhancing agents, and fluorescent compounds.

An “immunoconjugate” is a conjugate of an antibody with an atom,molecule, or a higher-ordered structure (e.g., with a liposome), atherapeutic agent, or a diagnostic agent. A “naked antibody” is anantibody that is not conjugated to any other agent.

A “naked antibody” is generally an entire antibody that is notconjugated to a therapeutic agent. This is so because the Fc portion ofthe antibody molecule provides effector functions, such as complementfixation and ADCC (antibody dependent cell cytotoxicity) that setmechanisms into action that may result in cell lysis. However, it ispossible that the Fc portion is not required for therapeutic function,with other mechanisms, such as apoptosis, coming into play. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric, humanized or humanantibodies.

As used herein, the term “antibody fusion protein” is a recombinantlyproduced antigen-binding molecule in which an antibody or antibodyfragment is linked to another protein or peptide, such as the same ordifferent antibody or antibody fragment or a DDD or AD peptide (of theDOCK-AND-LOCK™ complexes described below). The fusion protein maycomprise a single antibody component, a multivalent or multispecificcombination of different antibody components or multiple copies of thesame antibody component. The fusion protein may additionally comprise anantibody or an antibody fragment and a therapeutic agent. Examples oftherapeutic agents suitable for such fusion proteins includeimmunomodulators and toxins. One preferred toxin comprises aribonuclease (RNase), preferably a recombinant RNase.

A “multispecific antibody” is an antibody that can bind simultaneouslyto at least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. A “multivalent antibody” is anantibody that can bind simultaneously to at least two targets that areof the same or different structure. Valency indicates how many bindingarms or sites the antibody has to a single antigen or epitope; i.e.,monovalent, bivalent, trivalent or multivalent. The multivalency of theantibody means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen. Specificity indicates how many antigens or epitopes an antibodyis able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Multispecific, multivalent antibodiesare constructs that have more than one binding site of differentspecificity.

A “bispecific antibody” is an antibody that can bind simultaneously totwo targets which are of different structure. Bispecific antibodies(bsAb) and bispecific antibody fragments (bsFab) may have at least onearm that specifically binds to, for example, a B cell, T cell, myeloid-,plasma-, and mast-cell antigen or epitope and at least one other armthat specifically binds to a targetable conjugate that bears atherapeutic or diagnostic agent. A variety of bispecific antibodies canbe produced using molecular engineering. Included herein are bispecificantibodies that target a cancer-associated antigen and also animmunotherapeutic T cell, such as CD3-T cells.

The term “direct cytotoxicity” refers to the ability of an agent toinhibit the proliferation or induce the apoptosis of a cell grown in anoptimized culture medium in which only the agent and the cell arepresent.

Preparation of Monoclonal Antibodies

The compositions, formulations and methods described herein may includemonoclonal antibodies. Rodent monoclonal antibodies to specific antigensmay be obtained by methods known to those skilled in the art. (See,e.g., Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al.(eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (JohnWiley & Sons 1991)). General techniques for cloning murineimmunoglobulin variable domains have been disclosed, for example, by thepublication of Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833(1989).

Chimeric Antibodies

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Techniques forconstructing chimeric antibodies are well known to those of skill in theart. As an example, Leung et al., Hybridoma 13:469 (1994), disclose howthey produced an LL2 chimera by combining DNA sequences encoding theV_(k) and V_(H) domains of LL2 monoclonal antibody, an anti-CD22antibody, with respective human and IgG₁ constant region domains. Thispublication also provides the nucleotide sequences of the LL2 light andheavy chain variable regions, V_(k) and V_(H), respectively.

Humanized Antibodies

A chimeric monoclonal antibody can be humanized by replacing thesequences of the murine FR in the variable domains of the chimericantibody with one or more different human FR. Specifically, mouse CDRsare transferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more some human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. (See, e.g., Tempest et al., Biotechnology 9:266 (1991)and Verhoeyen et al., Science 239: 1534 (1988)). Techniques forproducing humanized antibodies are disclosed, for example, by Jones etal., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323 (1988),Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437(1992), and Singer et al., J. Immun. 150: 2844 (1993).

Human Antibodies

A fully human antibody can be obtained from a transgenic non-humananimal. (See, e.g., Mendez et al., Nature Genetics, 15: 146-156, 1997;U.S. Pat. No. 5,633,425.) Methods for producing fully human antibodiesusing either combinatorial approaches or transgenic animals transformedwith human immunoglobulin loci are known in the art (e.g., Mancini etal., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb.Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr.Opin. Pharmacol. 3:544-50; each incorporated herein by reference). Suchfully human antibodies are expected to exhibit even fewer side effectsthan chimeric or humanized antibodies and to function in vivo asessentially endogenous human antibodies. In certain embodiments, theclaimed methods and procedures may utilize human antibodies produced bysuch techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds),edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22, incorporated herein by reference). The final Fabfragments were digested with restriction endonucleases and inserted intothe bacteriophage genome to make the phage display library. Suchlibraries may be screened by standard phage display methods. The skilledartisan will realize that this technique is exemplary only and any knownmethod for making and screening human antibodies or antibody fragmentsby phage display may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.). In the XenoMouse® and similar animals, the mouse antibody geneshave been inactivated and replaced by functional human antibody genes,while the remainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding Vκ (variable light chain) and V_(H) (variable heavychain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of an antibody from a cell thatexpresses a murine antibody can be cloned by PCR amplification andsequenced. To confirm their authenticity, the cloned V_(L) and V_(H)genes can be expressed in cell culture as a chimeric Ab as described byOrlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based onthe V gene sequences, a humanized antibody can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine antibody by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The Vκ sequence for the antibody may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the Vκ andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human antibody. Alternatively, the Vκ andV_(H) expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:86) and veltuzumab (SEQ IDNO:85).

Veltuzumab Heavy Chain Constant Region Sequence (SEQ ID NO:85)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Rituximab Heavy Chain Constant Region Sequence (SEQ ID NO:86)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The Glm1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of Glm3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Known Antibodies

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art. For example,therapeutic use of anti-B cell antibodies, such as anti-CD22 antibodies,may be supplemented with one or more antibodies against otherdisease-associated antigens. Antibodies of use may be commerciallyobtained from a number of known sources. For example, a variety ofantibody secreting hybridoma lines are available from the American TypeCulture Collection (ATCC, Manassas, Va.). A large number of antibodiesagainst various disease targets, including but not limited totumor-associated antigens, have been deposited at the ATCC and/or havepublished variable region sequences and are available for use in theclaimed methods and compositions. See, e.g., U.S. Pat. Nos. 7,312,318;7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509; 7,049,060;7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498;7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852; 6,989,241;6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813; 6,956,107;6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645;6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681;6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594; 6,881,405;6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062; 6,861,511;6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780;6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711;6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981; 6,730,307;6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908; 6,689,607;6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734; 6,673,344;6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294;6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572,856;6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058; 6,528,625;6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930;6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173;6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310; 6,444,206;6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694;6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759;6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444;6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459;6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744; 6,129,914;6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440;5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136;5,587,459; 5,443,953, 5,525,338, the Examples section of each of whichis incorporated herein by reference. These are exemplary only and a widevariety of other antibodies and their hybridomas are known in the art.The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples sectionof each of which is incorporated herein by reference).

Antibodies of use may bind to various known antigens expressed in Bcells or T cells, including but not limited to BCL-1, BCL-2, BCL-6,CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14,CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40,CD40L, CD41a, CD43, CD45, CD55, CD56, CCD57, CD59, CD64, CD71, CD79a,CD79b, CD117, CD138, FMC-7 and HLA-DR.

Particular antibodies that may be of use for therapy of cancer withinthe scope of the claimed methods and compositions include, but are notlimited to, LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), RS7(anti-epithelial glycoprotein-1 (EGP-1)), PAM4 and KC4 (bothanti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known asCD66e), MN-15 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu31 (an anti-alpha-fetoprotein), TAG-72 (e.g., CC49), R1 (anti-IGF-1R),Tn, J591 or HuJ591 (anti-PSMA (prostate-specific membrane antigen),AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonicanhydrase IX), hL243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), GA101 (anti-CD20; obinutuzumab) and trastuzumab(anti-ErbB2). Such antibodies are known in the art (e.g., U.S. Pat. Nos.5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104;6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084;7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318;7,585,491; 7,612,180; 7,642,239; and U.S. Patent Application Publ. No.20040202666 (now abandoned); 20050271671; and 20060193865; the Examplessection of each incorporated herein by reference.) Specific knownantibodies of use include hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S.Pat. No. 7,251,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat.No. 7,300,655), hLL1 (U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No.7,074,403), hMu-9 (U.S. Pat. No. 7,387,773), hL243 (U.S. Pat. No.7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15 (U.S. Pat. No.7,541,440), hR1 (U.S. patent application Ser. No. 12/772,645), hRS7(U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No. 7,541,440),AB-PG1-XG1-026 (U.S. patent application Ser. No. 11/983,372, depositedas ATCC PTA-4405 and PTA-4406) and D2/B (WO 2009/130575), the text ofeach recited patent or application is incorporated herein by referencewith respect to the Figures and Examples sections.

Other antibodies of use for therapy of immune dysregulatory orautoimmune disease include, but are not limited to, anti-B-cellantibodies such as veltuzumab, epratuzumab, milatuzumab or hL243;tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25); daclizumab(anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3 receptor);OKT3 (anti-CDR3); anti-CD40L (UCB, Brussels, Belgium); natalizumab(anti-α4 integrin) and omalizumab (anti-IgE).

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab)₂, Fab′, Fab, Fv, scFv and the like. Otherantibody fragments include, but are not limited to: the F(ab′)₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab′ fragments, which can be generated by reducingdisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fab′expression libraries can be constructed (Huse et al., 1989, Science,246:1274-1281) to allow rapid and easy identification of monoclonal Fab′fragments with the desired specificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredisclosed in U.S. Pat. Nos. 4,704,692, 4,946,778, R. Raag and M.Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird andB. W. Walker, “Single Chain Antibody Variable Regions,” TIBTECH, Vol 9:132-137 (1991).

An antibody fragment can be prepared by known methods, for example, asdisclosed by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 andreferences contained therein. Also, see Nisonoff et al., Arch Biochem.Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman etal., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967),and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

A single complementarity-determining region (CDR) is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. (See, e.g., Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780).

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;Hwang and Foote, 2005, Methods 36:3-10; Clark, 2000, Immunol Today21:397-402; J Immunol 1976 117:1056-60; Ellison et al., 1982, Nucl AcidsRes 13:4071-79; Stickler et al., 2011, Genes and Immunity 12:213-21).

Multispecific and Multivalent Antibodies

Methods for producing bispecific antibodies include engineeredrecombinant antibodies which have additional cysteine residues so thatthey crosslink more strongly than the more common immunoglobulinisotypes. (See, e.g., FitzGerald et al, Protein Eng. 10(10):1221-1225,(1997)). Another approach is to engineer recombinant fusion proteinslinking two or more different single-chain antibody or antibody fragmentsegments with the needed dual specificities. (See, e.g., Coloma et al.,Nature Biotech. 15:159-163, (1997)). A variety of bispecific antibodiescan be produced using molecular engineering. In one form, the bispecificantibody may consist of, for example, an scFv with a single binding sitefor one antigen and a Fab fragment with a single binding site for asecond antigen. In another form, the bispecific antibody may consist of,for example, an IgG with two binding sites for one antigen and two scFvwith two binding sites for a second antigen. In alternative embodiments,multispecific and/or multivalent antibodies may be produced asDOCK-AND-LOCK™ (DNL™) complexes as described below.

In certain embodiments, one or more anti-B-cell antibodies may beadministered to a patient as part of a combination of antibodies.Bispecific antibodies are preferred to administration of combinations ofseparate antibodies, due to cost and convenience. However, wherecombinations of separate antibodies provide improved safety or efficacy,the combination may be utilized. The antibodies may bind to differentepitopes of the same antigen or to different antigens. Preferably, theantigens are selected from the group consisting of BCL-1, BCL-2, BCL-6,CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14,CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40,CD40L, CD41a, CD43, CD45, CD55, CD56, CCD57, CD59, CD64, CD71, CD79a,CD79b, CD117, CD138, FMC-7 and HLA-DR. However, antibodies against otherantigens of use for therapy of cancer, autoimmune diseases or immunedysfunction diseases are known in the art, as discussed below, andantibodies against any such disease-associated antigen known in the artmay be utilized.

DOCK-AND-LOCK™ (DNL™)

In preferred embodiments, a bivalent or multivalent antibody is formedas a DOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examples section ofeach of which is incorporated herein by reference.) Generally, thetechnique takes advantage of the specific and high-affinity bindinginteractions that occur between a dimerization and docking domain (DDD)sequence of the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and an anchor domain (AD) sequence derived from any of a varietyof AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wongand Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and ADpeptides may be attached to any protein, peptide or other molecule.Because the DDD sequences spontaneously dimerize and bind to the ADsequence, the technique allows the formation of complexes between anyselected molecules that may be attached to DDD or AD sequences.

Although the standard DNL™ complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL™complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL™ complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα or RIIβ (Newlon et al., Nat. Struct.Biol. 1999; 6:222). As discussed below, similar portions of the aminoacid sequences of other regulatory subunits are involved in dimerizationand docking, each located at or near the N-terminal end of theregulatory subunit. Binding of cAMP to the R subunits leads to therelease of active catalytic subunits for a broad spectrum ofserine/threonine kinase activities, which are oriented toward selectedsubstrates through the compartmentalization of PKA via its docking withAKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL™complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL™constructs of different stoichiometry may be produced and used (see,e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL™construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL™ constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL™ complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RIβ(SEQ ID NO: 9) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILAPKA RIIα (SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:12 to SEQ ID NO:31 below. The skilled artisan will realizethat an almost unlimited number of alternative species within the genusof DDD moieties can be constructed by standard techniques, for exampleusing a commercial peptide synthesizer or well known site-directedmutagenesis techniques. The effect of the amino acid substitutions on ADmoiety binding may also be readily determined by standard bindingassays, for example as disclosed in Alto et al. (2003, Proc Natl AcadSci USA 100:4445-50).

TABLE 2 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1).Consensus sequence disclosed as SEQ ID NO: 87. S H I Q I P P G L T E L LQ G Y T V E V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F TR L R E A R A N N E D L D S K K D L K L I I I V V V

(SEQ ID NO: 12) THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 13) SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 14) SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 15) SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 16) SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 17) SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 18) SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 19) SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 20) SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 21) SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 22) SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 23) SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 24) SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 25) SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA(SEQ ID NO: 26) SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA(SEQ ID NO: 27) SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA(SEQ ID NO: 28) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA(SEQ ID NO: 29) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA(SEQ ID NO: 30) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA(SEQ ID NO: 31) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 2 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

TABLE 3 Conservative Amino Acid Substitutions in AD1 (SEQ ID NO: 3).Consensus sequence disclosed as SEQ ID NO: 88. Q I E Y L A K Q I V D N AI Q Q A N L D F I R N E Q N N L V T V I S V

(SEQ ID NO: 32) NIEYLAKQIVDNAIQQA (SEQ ID NO: 33) QLEYLAKQIVDNAIQQA(SEQ ID NO: 34) QVEYLAKQIVDNAIQQA (SEQ ID NO: 35) QIDYLAKQIVDNAIQQA(SEQ ID NO: 36) QIEFLAKQIVDNAIQQA (SEQ ID NO: 37) QIETLAKQIVDNAIQQA(SEQ ID NO: 38) QIESLAKQIVDNAIQQA (SEQ ID NO: 39) QIEYIAKQIVDNAIQQA(SEQ ID NO: 40) QIEYVAKQIVDNAIQQA (SEQ ID NO: 41) QIEYLARQIVDNAIQQA(SEQ ID NO: 42) QIEYLAKNIVDNAIQQA (SEQ ID NO: 43) QIEYLAKQIVENAIQQA(SEQ ID NO: 44) QIEYLAKQIVDQAIQQA (SEQ ID NO: 45) QIEYLAKQIVDNAINQA(SEQ ID NO: 46) QIEYLAKQIVDNAIQNA (SEQ ID NO: 47) QIEYLAKQIVDNAIQQL(SEQ ID NO: 48) QIEYLAKQIVDNAIQQI (SEQ ID NO: 49) QIEYLAKQIVDNAIQQV

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL™ constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA(SEQ ID NO: 52) QIEYHAKQIVDHAIHQA (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs

AKAP-KL (SEQ ID NO: 54) PLEYQAGLLVQNAIQQAI AKAP79 (SEQ ID NO: 55)LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 56) LIEEAASRIVDAVIEQVK

RI-Specific AKAPs

AKAPce (SEQ ID NO: 57) ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 58)LEQVANQLADQIIKEAT PV38 (SEQ ID NO: 59) FEELAWKIAKMIWSDVF

Dual-Specificity AKAPs

AKAP7 (SEQ ID NO: 60) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 61)TAEEVSARIVQVVTAEAV DAKAP 1 (SEQ ID NO: 62) QIKQAAFQLISQVILEAT DAKAP2(SEQ ID NO: 63) LAWKIAKMIVSDVMQQ

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 65)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPIS QIEYLAKQIVDNAIQQA(SEQ ID NO: 3) AKAPIS-P QIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO:83) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 5. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 5 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1).Consensus sequence disclosed as SEQ ID NO: 89. S H I Q I P P G L T E L LQ G Y T V E V L R T N S I L A Q Q P P D L V E F A V E Y F T R L R E A RA N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL™ constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.). Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Affibodies and Fynomers

Certain alternative embodiments may utilize affibodies in place ofantibodies. Affibodies are commercially available from Affibody AB(Solna, Sweden). Affibodies are small proteins that function as antibodymimetics and are of use in binding target molecules. Affibodies weredeveloped by combinatorial engineering on an alpha helical proteinscaffold (Nord et al., 1995, Protein Eng 8:601-8; Nord et al., 1997, NatBiotechnol 15:772-77). The affibody design is based on a three helixbundle structure comprising the IgG binding domain of protein A (Nord etal., 1995; 1997). Affibodies with a wide range of binding affinities maybe produced by randomization of thirteen amino acids involved in the Fcbinding activity of the bacterial protein A (Nord et al., 1995; 1997).After randomization, the PCR amplified library was cloned into aphagemid vector for screening by phage display of the mutant proteins.The phage display library may be screened against any known antigen,using standard phage display screening techniques (e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, Quart. J. Nucl.Med. 43:159-162), in order to identify one or more affibodies againstthe target antigen.

A ¹⁷⁷Lu-labeled affibody specific for HER2/neu has been demonstrated totarget HER2-expressing xenografts in vivo (Tolmachev et al., 2007,Cancer Res 67:2773-82). Although renal toxicity due to accumulation ofthe low molecular weight radiolabeled compound was initially a problem,reversible binding to albumin reduced renal accumulation, enablingradionuclide-based therapy with labeled affibody (Id.).

The feasibility of using radiolabeled affibodies for in vivo tumorimaging has been recently demonstrated (Tolmachev et al., 2011,Bioconjugate Chem 22:894-902). A maleimide-derivatized NOTA wasconjugated to the anti-HER2 affibody and radiolabeled with ¹¹¹In (Id.).Administration to mice bearing the HER2-expressing DU-145 xenograft,followed by gamma camera imaging, allowed visualization of the xenograft(Id.).

Fynomers can also bind to target antigens with a similar affinity andspecificity to antibodies. Fynomers are based on the human Fyn SH3domain as a scaffold for assembly of binding molecules. The Fyn SH3domain is a fully human, 63 amino acid protein that can be produced inbacteria with high yields. Fynomers may be linked together to yield amultispecific binding protein with affinities for two or more differentantigen targets. Fynomers are commercially available from COVAGEN AG(Zurich, Switzerland).

The skilled artisan will realize that affibodies or fynomers may be usedas targeting molecules in the practice of the claimed methods andcompositions.

Pre-Targeting

Bispecific or multispecific antibodies may be utilized in pre-targetingtechniques. Pre-targeting is a multistep process originally developed toresolve the slow blood clearance of directly targeting antibodies, whichcontributes to undesirable toxicity to normal tissues such as bonemarrow. With pre-targeting, a radionuclide or other therapeutic agent isattached to a small delivery molecule (targetable construct) that iscleared within minutes from the blood. A pre-targeting bispecific ormultispecific antibody, which has binding sites for the targetableconstruct as well as a target antigen, is administered first, freeantibody is allowed to clear from circulation and then the targetableconstruct is administered.

Pre-targeting methods are disclosed, for example, in Goodwin et al.,U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988;Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl.Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988;Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl.Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991;Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl.Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991; U.S.Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702;7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702; and6,962,702, each incorporated herein by reference.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents.

Targetable Constructs

In certain embodiments, targetable construct peptides labeled with oneor more therapeutic or diagnostic agents for use in pre-targeting may beselected to bind to a bispecific antibody with one or more binding sitesfor a targetable construct peptide and one or more binding sites for atarget antigen associated with a disease or condition. Bispecificantibodies may be used in a pretargeting technique wherein the antibodymay be administered first to a subject. Sufficient time may be allowedfor the bispecific antibody to bind to a target antigen and for unboundantibody to clear from circulation. Then a targetable construct, such asa labeled peptide, may be administered to the subject and allowed tobind to the bispecific antibody and localize at the diseased cell ortissue.

Such targetable constructs can be of diverse structure and are selectednot only for the availability of an antibody or fragment that binds withhigh affinity to the targetable construct, but also for rapid in vivoclearance when used within the pre-targeting method and bispecificantibodies (bsAb) or multispecific antibodies. Hydrophobic agents arebest at eliciting strong immune responses, whereas hydrophilic agentsare preferred for rapid in vivo clearance. Thus, a balance betweenhydrophobic and hydrophilic character is established. This may beaccomplished, in part, by using hydrophilic chelating agents to offsetthe inherent hydrophobicity of many organic moieties. Also, sub-units ofthe targetable construct may be chosen which have opposite solutionproperties, for example, peptides, which contain amino acids, some ofwhich are hydrophobic and some of which are hydrophilic.

Peptides having as few as two amino acid residues, preferably two to tenresidues, may be used and may also be coupled to other moieties, such aschelating agents. The linker should be a low molecular weight conjugate,preferably having a molecular weight of less than 50,000 daltons, andadvantageously less than about 20,000 daltons, 10,000 daltons or 5,000daltons. More usually, the targetable construct peptide will have fouror more residues, such as the peptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂(SEQ ID NO: 90), wherein DOTA is1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid and HSG is thehistamine succinyl glycyl group. Alternatively, DOTA may be replaced byNOTA (1,4,7-triaza-cyclononane-1,4,7-triacetic acid), TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), NETA([2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl]-2-carbonylmethyl-amino]aceticacid) or other known chelating moieties. Chelating moieties may be used,for example, to bind to a therapeutic and or diagnostic radionuclide,paramagnetic ion or contrast agent.

The targetable construct may also comprise unnatural amino acids, e.g.,D-amino acids, in the backbone structure to increase the stability ofthe peptide in vivo. In alternative embodiments, other backbonestructures such as those constructed from non-natural amino acids orpeptoids may be used.

The peptides used as targetable constructs are conveniently synthesizedon an automated peptide synthesizer using a solid-phase support andstandard techniques of repetitive orthogonal deprotection and coupling.Free amino groups in the peptide, that are to be used later forconjugation of chelating moieties or other agents, are advantageouslyblocked with standard protecting groups such as a Boc group, whileN-terminal residues may be acetylated to increase serum stability. Suchprotecting groups are well known to the skilled artisan. See Greene andWuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons,N.Y.). When the peptides are prepared for later use within thebispecific antibody system, they are advantageously cleaved from theresins to generate the corresponding C-terminal amides, in order toinhibit in vivo carboxypeptidase activity. Exemplary methods of peptidesynthesis are disclosed in the Examples below.

Where pretargeting with bispecific antibodies is used, the antibody willcontain a first binding site for an antigen produced by or associatedwith a target tissue and a second binding site for a hapten on thetargetable construct. Exemplary haptens include, but are not limited to,HSG and In-DTPA. Antibodies raised to the HSG hapten are known (e.g. 679antibody) and can be easily incorporated into the appropriate bispecificantibody (see, e.g., U.S. Pat. Nos. 6,962,702; 7,138,103 and 7,300,644,incorporated herein by reference with respect to the Examples sections).However, other haptens and antibodies that bind to them are known in theart and may be used, such as In-DTPA and the 734 antibody (e.g., U.S.Pat. No. 7,534,431, the Examples section incorporated herein byreference).

Preparation of Immunoconjugates

In preferred embodiments, a therapeutic or diagnostic agent may becovalently attached to an antibody or antibody fragment to form animmunoconjugate. Where the immunoconjugate is to be administered inconcentrated form by subcutaneous, intramuscular or transdermaldelivery, the skilled artisan will realize that only non-cytotoxicagents may be conjugated to the antibody. Where a second antibody orfragment thereof is administered by a different route, such asintravenously, either before, simultaneously with or after thesubcutaneous, intramuscular or transdermal delivery, then the type ofdiagnostic or therapeutic agent that may be conjugated to the secondantibody or fragment thereof is not so limited, and may comprise anydiagnostic or therapeutic agent known in the art, including cytotoxicagents.

In some embodiments, a diagnostic and/or therapeutic agent may beattached to an antibody or fragment thereof via a carrier moiety.Carrier moieties may be attached, for example to reduced SH groupsand/or to carbohydrate side chains. A carrier moiety can be attached atthe hinge region of a reduced antibody component via disulfide bondformation. Alternatively, such agents can be attached using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the carrier moiety canbe conjugated via a carbohydrate moiety in the Fc region of theantibody.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component of theimmunoconjugate is an antibody fragment. However, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofa full length antibody or antibody fragment. See, for example, Leung etal., J. Immunol. 154: 5919 (1995); U.S. Pat. Nos. 5,443,953 and6,254,868, the Examples section of which is incorporated herein byreference. The engineered carbohydrate moiety is used to attach thetherapeutic or diagnostic agent.

An alternative method for attaching carrier moieties to a targetingmolecule involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.). For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.).

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.). Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.). An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.). The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.). Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.). These and other known clickchemistry reactions may be used to attach carrier moieties to antibodiesin vitro.

Agard et al. (2004, J Am Chem Soc 126:15046-47) demonstrated that arecombinant glycoprotein expressed in CHO cells in the presence ofperacetylated N-azidoacetylmannosamine resulted in the bioincorporationof the corresponding N-azidoacetyl sialic acid in the carbohydrates ofthe glycoprotein. The azido-derivatized glycoprotein reactedspecifically with a biotinylated cyclooctyne to form a biotinylatedglycoprotein, while control glycoprotein without the azido moietyremained unlabeled (Id.). Laughlin et al. (2008, Science 320:664-667)used a similar technique to metabolically label cell-surface glycans inzebrafish embryos incubated with peracetylatedN-azidoacetylgalactosamine. The azido-derivatized glycans reacted withdifluorinated cyclooctyne (DIFO) reagents to allow visualization ofglycans in vivo.

The Diels-Alder reaction has also been used for in vivo labeling ofmolecules. Rossin et al. (2010, Angew Chem Int Ed 49:3375-78) reported a52% yield in vivo between a tumor-localized anti-TAG72 (CC49) antibodycarrying a trans-cyclooctene (TCO) reactive moiety and an ¹¹¹In-labeledtetrazine DOTA derivative. The TCO-labeled CC49 antibody wasadministered to mice bearing colon cancer xenografts, followed 1 daylater by injection of ¹¹¹In-labeled tetrazine probe (Id.). The reactionof radiolabeled probe with tumor localized antibody resulted inpronounced radioactivity localization in the tumor, as demonstrated bySPECT imaging of live mice three hours after injection of radiolabeledprobe, with a tumor-to-muscle ratio of 13:1 (Id.). The results confirmedthe in vivo chemical reaction of the TCO and tetrazine-labeledmolecules.

Antibody labeling techniques using biological incorporation of labelingmoieties are further disclosed in U.S. Pat. No. 6,953,675 (the Examplessection of which is incorporated herein by reference). Such “landscaped”antibodies were prepared to have reactive ketone groups on glycosylatedsites. The method involved expressing cells transfected with anexpression vector encoding an antibody with one or more N-glycosylationsites in the CH1 or Vκ domain in culture medium comprising a ketonederivative of a saccharide or saccharide precursor. Ketone-derivatizedsaccharides or precursors included N-levulinoyl mannosamine andN-levulinoyl fucose. The landscaped antibodies were subsequently reactedwith agents comprising a ketone-reactive moiety, such as hydrazide,hydrazine, hydroxylamino or thiosemicarbazide groups, to form a labeledtargeting molecule. Exemplary agents attached to the landscapedantibodies included chelating agents like DTPA, large drug moleculessuch as doxorubicin-dextran, and acyl-hydrazide containing peptides. Thelandscaping technique is not limited to producing antibodies comprisingketone moieties, but may be used instead to introduce a click chemistryreactive group, such as a nitrone, an azide or a cyclooctyne, onto anantibody or other biological molecule.

Modifications of click chemistry reactions are suitable for use in vitroor in vivo. Reactive targeting molecule may be formed either by eitherchemical conjugation or by biological incorporation. The targetingmolecule, such as an antibody or antibody fragment, may be activatedwith an azido moiety, a substituted cyclooctyne or alkyne group, or anitrone moiety. Where the targeting molecule comprises an azido ornitrone group, the corresponding targetable construct will comprise asubstituted cyclooctyne or alkyne group, and vice versa. Such activatedmolecules may be made by metabolic incorporation in living cells, asdiscussed above.

Alternatively, methods of chemical conjugation of such moieties tobiomolecules are well known in the art, and any such known method may beutilized. General methods of immunoconjugate formation are disclosed,for example, in U.S. Pat. Nos. 4,699,784; 4,824,659; 5,525,338;5,677,427; 5,697,902; 5,716,595; 6,071,490; 6,187,284; 6,306,393;6,548,275; 6,653,104; 6,962,702; 7,033,572; 7,147,856; and 7,259,240,the Examples section of each incorporated herein by reference.

Therapeutic and Diagnostic Agents

In certain embodiments, the antibodies or fragments thereof may be usedin combination with one or more therapeutic and/or diagnostic agents.Where the agent is attached to an antibody or fragment thereof to beadministered by subcutaneous, intramuscular or transdermaladministration of a concentrated antibody formulation, then onlynon-cytotoxic agents are contemplated. Non-cytotoxic agents may include,without limitation, immunomodulators, cytokines (and their inhibitors),chemokines (and their inhibitors), tyrosine kinase inhibitors, growthfactors, hormones and certain enzymes (i.e., those that do not inducelocal necrosis), or their inhibitors. Where the agent is co-administeredeither before, simultaneously with or after the subcutaneous,intramuscular or transdermal antibody formulation, then cytotoxic agentsmay be utilized. An agent may be administered as an immunoconjugate witha second antibody or fragment thereof, or may be administered as a freeagent. The following discussion applies to both cytotoxic andnon-cytotoxic agents.

Therapeutic agents may be selected from the group consisting of aradionuclide, an immunomodulator, an anti-angiogenic agent, a cytokine,a chemokine, a growth factor, a hormone, a drug, a prodrug, an enzyme,an oligonucleotide, a pro-apoptotic agent, an interference RNA, aphotoactive therapeutic agent, a tyrosine kinase inhibitor, a Brutonkinase inhibitor, a sphingosine inhibitor, a cytotoxic agent, which maybe a chemotherapeutic agent or a toxin, and a combination thereof. Thedrugs of use may possess a pharmaceutical property selected from thegroup consisting of antimitotic, antikinase, alkylating, antimetabolite,antibiotic, alkaloid, anti-angiogenic, pro-apoptotic agents, andcombinations thereof.

Exemplary drugs may include, but are not limited to, 5-fluorouracil,aplidin, azaribine, anastrozole, anthracyclines, bendamustine,bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11),SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide,estramustine, epipodophyllotoxin, estrogen receptor binding agents,etoposide (VP16), etoposide glucuronide, etoposide phosphate,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, gemcitabine,hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide,leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, navelbine, nitrosourea, plicomycin, procarbazine, paclitaxel,pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen,taxol, temazolomide (an aqueous form of DTIC), transplatinum,thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracilmustard, vinorelbine, vinblastine, vincristine and vinca alkaloids.

Toxins may include ricin, abrin, alpha toxin, saporin, ribonuclease(RNase), e.g., onconase, DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.

Immunomodulators may be selected from a cytokine, a stem cell growthfactor, a lymphotoxin, a hematopoietic factor, a colony stimulatingfactor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and acombination thereof. Specifically useful are lymphotoxins such as tumornecrosis factor (TNF), hematopoietic factors, such as interleukin (IL),colony stimulating factor, such as granulocyte-colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF),interferon, such as interferons-α, -β, -λ, or -γ, and stem cell growthfactor, such as that designated “Si factor”. Included among thecytokines are growth hormones such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone; parathyroid hormone;thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoproteinhormones such as follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), and luteinizing hormone (LH); hepatic growth factor;prostaglandin, fibroblast growth factor; prolactin; placental lactogen,OB protein; tumor necrosis factor-α and -β; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-β; platelet-growth factor; transforminggrowth factors (TGFs) such as TGF-α and TGF-β; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-α, -β, -λ, and -γ; colony stimulatingfactors (CSFs) such as macrophage-CSF (M-CSF); interleukins (ILs) suchas IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23,IL-25, LIF, kit-ligand or FLT-3, angiostatin, thrombospondin,endostatin, tumor necrosis factor and lymphotoxin.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

Radioactive isotopes include, but are not limited to—¹¹¹In, ¹⁷⁷Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cb, ⁶⁷Cb, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb. The therapeutic radionuclidepreferably has a decay-energy in the range of 20 to 6,000 keV,preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.Maximum decay energies of useful beta-particle-emitting nuclides arepreferably 20-5,000 keV, more preferably 100-4,000 keV, and mostpreferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV. Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr ⁵⁹Fe ⁷⁵Se ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

A variety of tyrosine kinase inhibitors are known in the art and anysuch known therapeutic agent may be utilized. Exemplary tyrosine kinaseinhibitors include, but are not limited to canertinib, dasatinib,erlotinib, gefitinib, imatinib, lapatinib, leflunomide, nilotinib,pazopanib, semaxinib, sorafenib, sunitinib, sutent and vatalanib. Aspecific class of tyrosine kinase inhibitor is the Bruton tyrosinekinase inhibitor. Bruton tyrosine kinase (Btk) has a well-defined rolein B-cell development. Bruton kinase inhibitors include, but are notlimited to, PCI-32765 (ibrutinib), PCI-45292, GDC-0834, LFM-A13 andRN486.

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Joni et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Corticosteroid hormones can increase the effectiveness of otherchemotherapy agents, and consequently, they are frequently used incombination treatments. Prednisone and dexamethasone are examples ofcorticosteroid hormones.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-placenta growth factor (PlGF)peptides and antibodies, anti-vascular growth factor antibodies (such asanti-VEGF and anti-PlGF), anti-Flk-1 antibodies, anti-Flt-1 antibodiesand peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF(macrophage migration-inhibitory factor) antibodies, laminin peptides,fibronectin peptides, plasminogen activator inhibitors, tissuemetalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro-β,thrombospondin, 2-methoxyoestradiol, proliferin-related protein,carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,angiopoietin-2, interferon-alpha, interferon-lambda, herbimycin A,PNU145156E, 16K prolactin fragment, Linomide, thalidomide,pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin,angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, plateletfactor 4 or minocycline may be of use.

The therapeutic agent may comprise an oligonucleotide, such as a siRNA.The skilled artisan will realize that any siRNA or interference RNAspecies may be attached to an antibody or fragment thereof for deliveryto a targeted tissue. Many siRNA species against a wide variety oftargets are known in the art, and any such known siRNA may be utilizedin the claimed methods and compositions.

Known siRNA species of potential use include those specific forIKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S.Pat. No. 7,148,342); Bcl2 and EGFR (U.S. Pat. No. 7,541,453); CDC20(U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S. Pat. No.7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic anhydrase II (U.S.Pat. No. 7,579,457); complement component 3 (U.S. Pat. No. 7,582,746);interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Pat. No.7,592,443); survivin (U.S. Pat. No. 7,608,7070); superoxide dismutase 1(U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939);amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R(U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complementfactor B (U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), andapolipoprotein B (U.S. Pat. No. 7,795,421), the Examples section of eachreferenced patent incorporated herein by reference.

Additional siRNA species are available from known commercial sources,such as Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Minis Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either a siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools. Any such siRNA species may be delivered usingthe subject DNL complexes.

Exemplary siRNA species known in the art are listed in Table 6. AlthoughsiRNA is delivered as a double-stranded molecule, for simplicity onlythe sense strand sequences are shown in Table 6.

TABLE 6 Exemplary siRNA Sequences Target Sequence SEQ ID NO VEGF R2AATGCGGCGGTGGTGACAGTA SEQ ID NO: 91 VEGF R2 AAGCTCAGCACACAGAAAGACSEQ ID NO: 92 CXCR4 UAAAAUCUUCCUGCCCACCdTdT SEQ ID NO: 93 CXCR4GGAAGCUGUUGGCUGAAAAdTdT SEQ ID NO: 94 PPARC1 AAGACCAGCCUCUUUGCCCAGSEQ ID NO: 95 Dynamin 2 GGACCAGGCAGAAAACGAG SEQ ID NO: 96 CateninCUAUCAGGAUGACGCGG SEQ ID NO: 97 E1A binding proteinUGACACAGGCAGGCUUGACUU SEQ ID NO: 98 Plasminogen GGTGAAGAAGGGCGTCCAASEQ ID NO: 99 activator K-ras GATCCGTTGGAGCTGTTGGCGTAGTT SEQ ID NO: 100CAAGAGACTCGCCAACAGCTCCAACT TTTGGAAA Sortilin 1 AGGTGGTGTTAACAGCAGAGSEQ ID NO: 101 Apolipoprotein E AAGGTGGAGCAAGCGGTGGAG SEQ ID NO: 102Apolipoprotein E AAGGAGTTGAAGGCCGACAAA SEQ ID NO: 103 Bcl-XUAUGGAGCUGCAGAGGAUGdTdT SEQ ID NO: 104 Raf-1 TTTGAATATCTGTGCTGAGAACACASEQ ID NO: 105 GTTCTCAGCACAGATATTCTTTTT Heat shockAATGAGAAAAGCAAAAGGTGCCCTGTCTC SEQ ID NO: 106 transcription factor 2IGFBP3 AAUCAUCAUCAAGAAAGGGCA SEQ ID NO: 107 ThioredoxinAUGACUGUCAGGAUGUUGCdTdT SEQ ID NO: 108 CD44 GAACGAAUCCUGAAGACAUCUSEQ ID NO: 109 MMP14 AAGCCTGGCTACAGCAATATGCCTGTCTC SEQ ID NO: 110MAPKAPK2 UGACCAUCACCGAGUUUAUdTdT SEQ ID NO: 111 FGFR1AAGTCGGACGCAACAGAGAAA SEQ ID NO: 112 ERBB2 CUACCUUUCUACGGACGUGdTdTSEQ ID NO: 113 BCL2L1 CTGCCTAAGGCGGATTTGAAT SEQ ID NO: 114 ABL1TTAUUCCUUCUUCGGGAAGUC SEQ ID NO: 115 CEACAM1 AACCTTCTGGAACCCGCCCACSEQ ID NO: 116 CD9 GAGCATCTTCGAGCAAGAA SEQ ID NO: 117 CD151CATGTGGCACCGTTTGCCT SEQ ID NO: 118 Caspase 8 AACTACCAGAAAGGTATACCTSEQ ID NO: 119 BRCA1 UCACAGUGUCCUUUAUGUAdTdT SEQ ID NO: 120 p53GCAUGAACCGGAGGCCCAUTT SEQ ID NO: 121 CEACAM6 CCGGACAGTTCCATGTATASEQ ID NO: 122

The skilled artisan will realize that Table 6 represents a very smallsampling of the total number of siRNA species known in the art, and thatany such known siRNA may be utilized in the claimed methods andcompositions.

Diagnostic agents are preferably selected from the group consisting of aradionuclide, a radiological contrast agent, a paramagnetic ion, ametal, a fluorescent label, a chemiluminescent label, an ultrasoundcontrast agent and a photoactive agent. Such diagnostic agents are wellknown and any such known diagnostic agent may be used. Non-limitingexamples of diagnostic agents may include a radionuclide such as ¹⁸F,⁵²Fe, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y,⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd,³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br,⁷⁶Br, ^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters.

Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III).

Ultrasound contrast agents may comprise liposomes, such as gas filledliposomes. Radiopaque diagnostic agents may be selected from compounds,barium compounds, gallium compounds, and thallium compounds. A widevariety of fluorescent labels are known in the art, including but notlimited to fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.Chemiluminescent labels of use may include luminol, isoluminol, anaromatic acridinium ester, an imidazole, an acridinium salt or anoxalate ester.

Methods of Administration

The subject antibodies and immunoglobulins in general may be formulatedto obtain compositions that include one or more pharmaceuticallysuitable excipients, surfactants, polyols, buffers, salts, amino acids,or additional ingredients, or some combination of these. This can beaccomplished by known methods to prepare pharmaceutically usefuldosages, whereby the active ingredients (i.e., the labeled molecules)are combined in a mixture with one or more pharmaceutically suitableexcipients. Sterile phosphate-buffered saline is one example of apharmaceutically suitable excipient. Other suitable excipients are wellknown to those in the art. See, e.g., Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

The preferred route for administration of the compositions describedherein is parenteral injection, more preferably by subcutaneous,intramuscular or transdermal delivery. Other forms of parenteraladministration include intravenous, intraarterial, intralymphatic,intrathecal, intraocular, intracerebral, or intracavitary injection. Inparenteral administration, the compositions will be formulated in a unitdosage injectable form such as a solution, suspension or emulsion, inassociation with a pharmaceutically acceptable excipient. Suchexcipients are inherently nontoxic and nontherapeutic. Examples of suchexcipients are saline, Ringer's solution, dextrose solution and Hanks'solution. Nonaqueous excipients such as fixed oils and ethyl oleate mayalso be used. An alternative excipient is 5% dextrose in saline. Theexcipient may contain minor amounts of additives such as substances thatenhance isotonicity and chemical stability, including buffers andpreservatives.

Formulated compositions comprising antibodies can be used forsubcutaneous, intramuscular or transdermal administration. Compositionscan be presented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. Compositions can also take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the compositionscan be in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use.

The compositions may be administered in solution. The formulationthereof should be in a solution having a suitable pharmaceuticallyacceptable buffer such as phosphate, TRIS (hydroxymethyl)aminomethane-HCl or citrate and the like. Buffer concentrations shouldbe in the range of 1 to 100 mM. The formulated solution may also containa salt, such as sodium chloride or potassium chloride in a concentrationof 50 to 150 mM. An effective amount of a stabilizing agent such asmannitol, trehalose, sorbitol, glycerol, albumin, a globulin, adetergent, a gelatin, a protamine or a salt of protamine may also beincluded.

The dosage of an administered antibody for humans will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of antibody that is inthe range of from about 1 mg to 600 mg as a single infusion or single ormultiple injections, although a lower or higher dosage also may beadministered. Typically, it is desirable to provide the recipient with adosage that is in the range of from about 50 mg per square meter (m²) ofbody surface area or 70 to 85 mg of the antibody for the typical adult,although a lower or higher dosage also may be administered. Examples ofdosages of antibodies that may be administered to a human subject are 1to 1,000 mg, more preferably 1 to 70 mg, most preferably 1 to 20 mg,although higher or lower doses may be used. Dosages may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths, or more frequently, such as twice weekly or by continuousinfusion.

More recently, subcutaneous administration of veltuzumab has been givento NHL patients in 4 doses of 80, 160 or 320 mg, repeated every twoweeks (Negrea et al., 2011, Haematologica 96:567-73). Only occasional,mild to moderate and transient injection reactions were observed, withno other safety issues (Id.). The objective response rate (CR+CRu+PR)was 47%, with a CR/CRu (complete response) rate of 24% (Id.).Interestingly, the 80 mg dosage group showed the highest percentage ofobjective response (⅔, 67%), with one of three patients showing acomplete response (Id.). Four out of eight objective responses continuedfor 60 weeks (Id.). All serum samples evaluated for HAHA were negative(Id.). Although the low sample population reported in this studyprecludes any definitive conclusions on optimal dosing, it is apparentthat therapeutic response was observed at the lowest dosage tested (80mg).

In certain alternative embodiments, the antibody may be administered bytransdermal delivery. Different methods of transdermal delivery areknown in the art, such as by transdermal patches or by microneedledevices, and any such known method may be utilized. In an exemplaryembodiment, transdermal delivery may utilize a delivery device such asthe 3M hollow Microstructured Transdermal System (hMTS) for antibodybased therapeutics. The hMTS device comprises a 1 cm² microneedle arrayconsisting of 18 hollow microneedles that are 950 microns in length,which penetrate approximately 600-700 microns into the dermal layer ofthe skin where there is a high density of lymphatic channels. Aspring-loaded device forces the antibody composition from a fluidreservoir through the microneedles for delivery to the subject. Onlytransient erythema and edema at the injection site are observed (Burtonet al., 2011, Pharm Res 28:31-40). The hMTS device is not perceived as aneedle injector, resulting in improved patient compliance.

In alternative embodiments, transdermal delivery of peptides andproteins may be achieved by (1) coadministering with a synthetic peptidecomprising the amino acid sequence of ACSSSPSKHCG (SEQ ID NO:123) asreported by Chen et al. (Nat Biotechnol 2006; 24: 455-460) andCarmichael et al. (Pain 2010; 149:316-324); (2) coadministering witharginine-rich intracellular delivery peptides as reported by Wang et al.(BBRC 2006; 346: 758-767); (3) coadminstering with either AT1002(FCIGRLCG, SEQ ID NO:124) or Tat (GRKKRRNRRRCG, SEQ ID NO:125) asreported by Uchida et al. (Chem Pharm Bull 2011; 59:196); or (4) usingan adhesive transdermal patch as reported by Jurynczyk et al (Ann Neurol2010; 68:593-601). In addition, transdermal delivery of negativelycharged drugs may be facilitated by combining with the positivelycharged, pore-forming magainin peptide as reported by Kim et al. (Int JPharm 2008; 362:20-28).

In preferred embodiments where the antibody is administeredsubcutaneously, intramuscularly or transdermally in a concentratedformulation, the volume of administration is preferably limited to 3 mlor less, more preferably 2 ml or less, more preferably 1 ml or less. Theuse of concentrated antibody formulations allowing low volumesubcutaneous, intramuscular or transdermal administration is preferredto the use of more dilute antibody formulations that require specializeddevices and ingredients (e.g., hyaluronidase) for subcutaneousadministration of larger volumes of fluid, such as 10 ml or more. Thesubcutaneous, intramuscular or transdermal delivery may be administeredas a single administration to one skin site or alternatively may berepeated one or more times, or even given to more than one skin site inone therapeutic dosing session. However, the more concentrated theformulation, the lower the volume injected and the fewer injections willbe needed for each therapeutic dosing.

Methods of Use

In preferred embodiments, the concentrated antibodies are of use fortherapy of cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, blastoma, glioma, melanoma, sarcoma, and leukemiaor lymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g. epithelial squamouscell cancer), lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, neuroblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectalcancer, endometrial cancer or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, anal carcinoma, penile carcinoma, as well as head andneck cancer. The term “cancer” includes primary malignant cells ortumors (e.g., those whose cells have not migrated to sites in thesubject's body other than the site of the original malignancy or tumor)and secondary malignant cells or tumors (e.g., those arising frommetastasis, the migration of malignant cells or tumor cells to secondarysites that are different from the site of the original tumor).

Other examples of cancers or malignancies include, but are not limitedto: acute childhood lymphoblastic leukemia, acute lymphoblasticleukemia, acute lymphocytic leukemia, acute myeloid leukemia,adrenocortical carcinoma, adult (primary) hepatocellular cancer, adult(primary) liver cancer, adult acute lymphocytic leukemia, adult acutemyeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma,adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primaryliver cancer, adult soft tissue sarcoma, AIDS-related lymphoma,AIDS-related malignancies, anal cancer, astrocytoma, bile duct cancer,bladder cancer, bone cancer, brain stem glioma, brain tumors, breastcancer, cancer of the renal pelvis and ureter, central nervous system(primary) lymphoma, central nervous system lymphoma, cerebellarastrocytoma, cerebral astrocytoma, cervical cancer, childhood (primary)hepatocellular cancer, childhood (primary) liver cancer, childhood acutelymphoblastic leukemia, childhood acute myeloid leukemia, childhoodbrain stem glioma, childhood cerebellar astrocytoma, childhood cerebralastrocytoma, childhood extracranial germ cell tumors, childhoodHodgkin's disease, childhood Hodgkin's lymphoma, childhood hypothalamicand visual pathway glioma, childhood lymphoblastic leukemia, childhoodmedulloblastoma, childhood non-Hodgkin's lymphoma, childhood pineal andsupratentorial primitive neuroectodermal tumors, childhood primary livercancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma,childhood visual pathway and hypothalamic glioma, chronic lymphocyticleukemia, chronic myelogenous leukemia, colon cancer, cutaneous T-celllymphoma, endocrine pancreas islet cell carcinoma, endometrial cancer,ependymoma, epithelial cancer, esophageal cancer, Ewing's sarcoma andrelated tumors, exocrine pancreatic cancer, extracranial germ celltumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eyecancer, female breast cancer, Gaucher's disease, gallbladder cancer,gastric cancer, gastrointestinal carcinoid tumor, gastrointestinaltumors, germ cell tumors, gestational trophoblastic tumor, hairy cellleukemia, head and neck cancer, hepatocellular cancer, Hodgkin'sdisease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngealcancer, intestinal cancers, intraocular melanoma, islet cell carcinoma,islet cell pancreatic cancer, Kaposi's sarcoma, kidney cancer, laryngealcancer, lip and oral cavity cancer, liver cancer, lung cancer,lymphoproliferative disorders, macroglobulinemia, male breast cancer,malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma,mesothelioma, metastatic occult primary squamous neck cancer, metastaticprimary squamous neck cancer, metastatic squamous neck cancer, multiplemyeloma, multiple myeloma/plasma cell neoplasm, myelodysplasticsyndrome, myelogenous leukemia, myeloid leukemia, myeloproliferativedisorders, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, non-Hodgkin's lymphoma during pregnancy,nonmelanoma skin cancer, non-small cell lung cancer, occult primarymetastatic squamous neck cancer, oropharyngeal cancer, osteo-/malignantfibrous sarcoma, osteosarcoma/malignant fibrous hi stiocytoma,osteosarcoma/malignant fibrous histiocytoma of bone, ovarian epithelialcancer, ovarian germ cell tumor, ovarian low malignant potential tumor,pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, penilecancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm/multiplemyeloma, primary central nervous system lymphoma, primary liver cancer,prostate cancer, rectal cancer, renal cell cancer, renal pelvis andureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,sarcoidosis sarcomas, Sezary syndrome, skin cancer, small cell lungcancer, small intestine cancer, soft tissue sarcoma, squamous neckcancer, stomach cancer, supratentorial primitive neuroectodermal andpineal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroidcancer, transitional cell cancer of the renal pelvis and ureter,transitional renal pelvis and ureter cancer, trophoblastic tumors,ureter and renal pelvis cell cancer, urethral cancer, uterine cancer,uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma,vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used todetect or treat malignant or premalignant conditions. Such uses areindicated in conditions known or suspected of preceding progression toneoplasia or cancer, in particular, where non-neoplastic cell growthconsisting of hyperplasia, metaplasia, or most particularly, dysplasiahas occurred (for review of such abnormal growth conditions, see Robbinsand Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia,pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be detected include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be detected and/or treatedinclude, but are not limited to, benign dysproliferative disorders(e.g., benign tumors, fibrocystic conditions, tissue hypertrophy,intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia,keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solarkeratosis.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

The exemplary conditions listed above that may be treated are notlimiting. The skilled artisan will be aware that antibodies or antibodyfragments are known for a wide variety of conditions, such as autoimmunedisease, graft-versus-host-disease, organ transplant rejection,cardiovascular disease, neurodegenerative disease, metabolic disease,cancer, infectious disease and hyperproliferative disease.

Exemplary autoimmune diseases include acute idiopathic thrombocytopenicpurpura, chronic immune thrombocytopenia, dermatomyositis, Sydenham'schorea, myasthenia gravis, systemic lupus erythematosus, lupusnephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid,pemphigus vulgaris, juvenile diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, ANCA-associated vasculitides, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis and fibrosingalveolitis.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one concentrated antibody or fragment thereof as described herein.A device capable of delivering the kit components by injection, forexample, a syringe for subcutaneous injection, may be included. Wheretransdermal administration is used, a delivery device such as hollowmicroneedle delivery device may be included in the kit. Exemplarytransdermal delivery devices are known in the art, such as 3M's hollowMicrostructured Transdermal System (hMTS), and any such known device maybe used.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents.Alternatively, the concentrated antibody may be delivered and stored asa liquid formulation. Other containers that may be used include, but arenot limited to, a pouch, tray, box, tube, or the like. Kit componentsmay be packaged and maintained sterilely within the containers. Anothercomponent that can be included is instructions to a person using a kitfor its use.

EXAMPLES Example 1 Epratuzumab-Induced Trogocytosis of BCR-ResponseModulating Proteins Ex Vivo

The humanized anti-CD22 antibody, epratuzumab, has demonstratedtherapeutic activity in clinical trials of patients with non-Hodgkinlymphoma (NHL), acute lymphoblastic leukemia, primary Sjögren'ssyndrome, and systemic lupus erythematosus (SLE). Thus, epratuzumaboffers a promising option for CD22-targeted immunotherapy of B-celllymphomas and autoimmune diseases. However, its mechanism of action(MOA) remains incompletely understood to-date. Because epratuzumab hasmodest, but significant, antibody-dependent cell-mediated cytotoxicityand negligible complement-dependent cytotoxicity when evaluated invitro, and its moderate depletion of circulating B cells in patients(35% on average) may be overestimated due to use of CD19⁺ cells tomeasure B cells by flow cytometry (discussed below), the therapeuticaction of epratuzumab in vivo may not result from B-cell depletion. Weinvestigated whether ligation of epratuzumab to CD22 could modulateother surface molecules on B cells. In particular, we focused on thosesurface molecules involved in regulating antigen-specific B-cellreceptor (BCR) signaling, since modulation of such molecules may lead toaltered B-cell functions that ultimately mitigate symptoms of autoimmuneor other diseases. With regard to its function of killing malignant Bcells expressing CD22, our studies have shown that these effects aremore related to the BCR signaling pathway than effector-cell function.

Here we report for the first time that epratuzumab induces a substantialreduction of CD22, along with CD19, CD21, CD20, and CD79b, on thesurface of B cells in peripheral blood mononuclear cells (PBMCs)obtained from normal donors or lupus patients, and three NHL Burkittcell lines (Daudi, Raji, and Ramos) spiked into normal PBMCs. Theintriguing observation that only CD22, but not other surface markers,was appreciably decreased by epratuzumab in isolated NHL cells promptedus to assess the role of FcγR-bearing effector cells, with the findingthat epratuzumab effectively mediates trogocytosis [a process wherebycells binding to antigen-presenting cells extract surface molecules fromthese cells and express them on their own surface] of multiple surfaceproteins from B cells to monocytes, NK cells, and neutrophils. Thismechanism of action may explain the limited effectiveness of high dosesof epratuzumab compared to lower doses in patients with SLE.

Peripheral blood mononuclear cells (PBMCs) obtained from healthy donorswere incubated overnight (16-24 h) with 10 μg/mL of either epratuzumabor an isotype control mAb (hMN-14) and the relative levels of variousantigens on the surface of the B cells were analyzed by flow cytometry.PBMCs from heparinized whole blood of normal donors were isolated bydensity gradient centrifugation on UNI-SEP tubes (Novamed Ltd, Israel).PBMCs were reconstituted in RPMI media supplemented with 10% heatinactivated fetal bovine serum and plated at a cell density of1.5×10⁶/mL in non-tissue culture treated 48-well plates. Epratuzumab orhMN-14 were added to triplicate wells at a final concentration of 10μg/mL and incubated overnight (16-20 h) before staining withfluorescent-labeled primary antibodies (Biolegend) following themanufacturers suggested protocols. Stained cells were analyzed by flowcytometry on a FACSCALIBUR® (BD Biosciences) using Flowjo (V7.6.5)software. Initially, the lymphocyte population was gated by side vs.forward scattering, and B cells were further gated from this populationwith the CD19 signal. The mean fluorescence intensity (MFI), obtainedwith fluorochrome-conjugated antibodies to various cell surfaceantigens, on the gated B cells was calculated following treatment withepratuzumab, hMN-14 or without antibody. PBMCs from 16 healthy donorswere assessed in various experiments.

Treatment with the control mAb (hMN-14) did not affect the levels of anyof the tested proteins and resulted in MFI measurements that were verysimilar to untreated samples. Alternatively, epratuzumab significantlyreduced the levels of key BCR-regulating proteins, including CD22, CD19,CD21 and CD79b, which were reduced to 10, 50, 52 and 70%, respectively,of the level of untreated or control mAb (FIG. 1). CD20 (82%) and CD62L(73%) also were reduced, but to a lesser extent. Other surface proteinsincluding CD27 (on CD27⁺ B cells), CD40, CD44, CD45, β7 integrin andLFA-1 (CD11a and CD18) were affected minimally (<10% change) byepratuzumab. CD27⁻ naive B cells were more responsive to epratuzumabcompared to CD27⁺ memory B cells, as shown with PBMCs as shown for CD19from 3 different healthy donors (FIG. 2). CD22, CD21 and CD79b were alsoreduced to a greater extent on CD27⁻ cells (FIG. 3). The effect wasessentially complete within a few hours. The reductions in surface CD19and CD21 were not significantly different following 2-h or overnighttreatment (FIG. 4).

Example 2 Effect of Various B Cell-Targeting Antibodies

We investigated whether the reciprocal effect might occur, whereby a mAbto CD19 (mAb hA19) could reduce the surface level of CD22 from B cellswithin PBMCs. However, hA19 had no effect on the level of CD22 (FIG. 5).We were unable to determine the level of CD19 following treatment withhA19 because its binding blocked detection with anti-CD19. TheCD20-targeting mAbs rituximab and veltuzumab each diminished CD19, CD21and CD79b to a greater extent than epratuzumab (FIG. 6). Rituximab alsoreduced CD22, but to a lesser extent than epratuzumab. Notably,rituximab and veltuzumab (at 10 μg/mL) reduced the B cell count by 50%,and 40%, where epratuzumab did not cause significant B cell depletion,either at 10 μg/mL or 1 mg/mL.

TABLE 7 Comparison of epratuzumab with other humanized antibodiestargeting different antigens on B cells. Target mAb CD19 CD20 CD21 CD22CD74 CD22 epratuzumab 30-60% (↓) 10-30% (↓) 30-60% (↓) >60% (↓) 10-30%(↑) CD20 veltuzumab >60% (↓) nd >60% (↓) 10-30% (↓) 30-60% (↑) CD19 hA19nd <10% 10-30% (↓) <10% 30-60% (↑) CD74 milatuzumab <10% <10% 10-30% (↓)nd nd (↓), decreased; (↑), increased; nd, not determined

Example 3 Dose-Dependent Trogocytosis with Epratuzumab

The effect of epratuzumab on the cell surface levels of CD19, CD21, CD22and CD79b was compared using the standard (10 μg/mL) concentration witha 100-fold higher concentration (1 mg/mL). An additional treatmentincluded 10 μg/mL epratuzumab combined with 1 mg/mL hMN-14. Compared tothe lower concentration of epratuzumab (10 μg/mL), the higherconcentration (1 mg/mL) resulted in significantly (P<0.02) lessreduction in CD22, CD19, CD21 and CD79b (FIG. 7). Competition with highconcentration (1 mg/mL) hMN-14 significantly (P<0.003) reduced theeffect of epratuzumab (10 μg/mL) on CD22 and CD19, but to a lesserextent than high-dose epratuzumab. A titration experiment, where normalPBMCs were incubated overnight with epratuzumab at concentrationsranging from 0.1-1000 μg/mL, confirmed that doses approaching 1 mg/mLdampened the effect (FIG. 8, donor N13, dashed curves). A secondtitration covering 8 logs (1 ng/mL-10 mg/mL) produced a classic U-shapedcurve with substantial dampening at concentrations lower than 10 ng/mLor greater than 1 mg/mL (FIG. 8, donor N14, solid curves). The reductionof both CD22 and CD19 on B cells within PBMCs was similar over a wideconcentration range (10 ng/mL-100 μg/mL) of epratuzumab.

Example 4 The Fc is Required for Trogocytosis

An F(ab′)₂ fragment of epratuzumab, which was prepared by pepsindigestion, reduced CD22 moderately (45% control), compared to the fullIgG (10% control), and had no effect on CD19, CD21 and CD79b (FIG. 9).The loss of CD22 can be attributed to internalization of theantibody/antigen complex, which is a well established phenomenonassociated with epratuzumab, and not due to trogocytosis. That CD19,CD21 and CD79 are not affected by the F(ab′)₂ indicates that notrogocytosis is induced by the Fc-lacking antibody fragment. A similarfinding was observed when PBMCs from lupus patients were used instead offrom healthy donors (Example 10).

Example 5 Effector Cells are Required for Epratuzumab-InducedTrogocytosis

B cell lymphoma cell lines were used as “isolated B cells” that wereevaluated for epratuzumab induced trogocytosis. In vitro, epratuzumabinduced an intermediate reduction (33% control) of CD22 on the surfaceof isolated Daudi Burkitt lymphoma cells, and did not affect the levelsof other markers (FIG. 10). In an ex vivo setting, where Daudi werespiked into PBMCs from a healthy donor, epratuzumab minimized CD22 (<5%control) and significantly (P<0.0001) reduced CD19 (28% control), CD21(40% control), CD79b (72% control) and surface IgM (73% control).Similar results were obtained with Raji lymphoma cells, where CD19, CD21and CD79b were diminished by epratuzumab only in the presence of PBMCs(FIG. 11). The addition of a crosslinking second antibody resulted inonly a modest reduction of CD19, CD21 and CD79b. That the effect onlywas observed in the presence of PBMCs, and it was not accomplished inthe presence of PBMCs with a F(ab′)₂ fragment (Example 4) or with acrosslinking second antibody in place of PBMCs, indicates that effectorcells bearing Fc receptors are involved in the epratuzumab-inducedtrogocytosis process.

Example 6 Monocytes, but not T Cells can Modulate Epratuzumab-InducedTrogocytosis

Combined, T cells and monocytes comprise approximately 70-80% of thetotal PBMCs. The ability of PBMC fractions, which were depleted ofeither T cells or monocytes using MACS separation technology (MiltenyiBiotec) with magnetically labeled microbeads in an LS or MS column, wereevaluated for epratuzumab-induced reduction of CD22 and CD19 on Daudiand normal B cells. For this experiment the ratio of total effectorcells to Daudi was held constant. Therefore, removal of a specific celltype resulted in increased numbers of the remaining cell types (FIG.12). Depletion of T cells was only 50% efficient; however, this resultedin a 10% increase in monocytes and other cell types. The T-cell-depletedPBMCs were significantly more active than total PBMCs, indicating that Tcells are not involved (FIG. 13). Indeed, purified T cells were notcapable of affecting the epratuzumab-induced reduction of CD19 or CD21on Daudi (FIG. 14). Conversely, depletion of monocytes, which was 99%efficient (FIG. 12), significantly dampened the reduction of both CD19and CD22 on either Daudi or B cells (FIG. 13), implicating theinvolvement of monocytes. That there was appreciable reduction of CD19with the monocyte-depleted PBMCs, suggests the participation ofadditional cell types. In a subsequent experiment, purified monocytes(94%, FIG. 15) induced a similar decrease in CD19 as the whole PBMCs,whereas the remaining monocyte-depleted PBMCs had minimal effect,comparable to the levels measured without effector cells (FIG. 16). Asimilar pattern was observed for CD22. This particular donor gaverelatively weak activity (25% reduction in CD19) compared to mostothers, where we have typically observed a 40-60% reduction in CD19.Nonetheless, the results support the key role of monocytes among PBMCs.

Example 7 Epratuzumab-Induced Trogocytosis with Monocytes

Trogocytosis involves the transfer of membrane components from one cellto another. To determine if the loss of surface antigen on B cells isdue to their transfer to effector cells (trogocytosis), Daudi cells weremixed with PBMCs (FIG. 17), purified monocytes (FIG. 18) ormonocyte-depleted PBMCs, and treated with epratuzumab or the isotypecontrol for 1 h. Daudi, monocyte and lymphocyte populations were gatedby foreword vs. side scattering. When mixed with Daudi cells and treatedwith epratuzumab, but not the isotype control mAb, purified monocytes(CD14 positive cells) stained positive for either CD22 (56.6% positive)and CD19 (52.4% positive), with 44% positive for both (FIG. 19).Treatment with an isotype control mAb resulted in only 1.6% doublepositive monocytes. The monocytes were further gated into CD14⁺⁺ (˜90%)and CD14⁺CD16⁺ (−10%) sub-populations (FIG. 17 and FIG. 18). TheCD14⁺CD16⁺ monocytes (FIG. 20A) exhibited more activity (66.4%CD19⁺CD22⁺) compared to the more abundant CD14⁺⁺ (31.4%) cells (FIG.20B). Even after only 1 h, CD19 and CD22 were specifically reduced fromDaudi cells when treated with epratuzumab in the presence of PBMCs orpurified monocytes (FIG. 21). These results demonstrate that CD19 andCD22 are transferred from Daudi cells to both populations of monocytes.

Example 8 Epratuzumab-Induced Trogocytosis with NK Cells

CD19 and CD22 were significantly reduced from Daudi cells inmonocyte-depleted PBMCs (FIG. 21), suggesting the involvement ofeffector cells in addition to monocytes. NK cells, which express FcγRIII(CD16), are identified among PBMCs by flow cytometry as CD14-CD16+ cellslocated in the lymphocyte (forward vs. side scatter) gate. Using theDaudi/PBMC and Daudi/monocyte-depleted PBMC mixtures from Example 7, thelymphocyte gate was further gated for CD14 and CD16 to identifyCD14⁻CD16⁺ NK cells (FIG. 22). NK cells potently acquired CD19 and CD22when either PBMCs (FIG. 23A) or monocyte-depleted PBMCs (FIG. 23B) weremixed with Daudi and epratuzumab. These results indicate that NK cellscan function in epratuzumab-induced trogocytosis.

Example 9 Epratuzumab-Induced Trogocytosis with Granulocytes

Granulocytes, or polymorphonuclear cells, which comprise mostlyneutrophils, are separated from the PBMCs during processing of wholeblood. Granulocytes, which express FcγRIII (CD16), were assessed fortheir ability to participate in trogocytosis when mixed with Daudi cellsand epratuzumab. Granulocytes were readily gated from the Daudi cells byside scattering and CD16 (FIG. 24). When mixed with Daudi cells andtreated with epratuzumab, but not the isotype control mAb, granulocytesstained positive for CD22 (30.4% positive), CD19 (40.9% positive) andCD79b (13.7% positive) (FIG. 25). Following the 1-h incubation, asignificant reduction on Daudi of each antigen indicates their transferfrom Daudi to granulocytes (FIG. 26).

TABLE 8 Trogocytosis of CD19 and CD22 from Daudi to monocytes, NK cellsand granulocytes following treatment with epratuzumab. Cells mAb % CD19⁺% CD22⁺ % CD19⁺CD22⁺ All epratuzumab 52.4 56.6 44.4 Monocytes hMN-1410.1 5.3 1.6 CD14⁺CD16⁺ epratuzumab 67.5 81.6 66.4 Monocytes hMN-14 4.36.7 2.3 CD14⁺⁺ epratuzumab 35.4 48.9 31.4 Monocytes hMN-14 2.1 2.6 0.5CD14⁻CD16⁺ epratuzumab 46.3 58.0 43.6 NK hMN-14 3.7 4.7 2.4 Granulocytesepratuzumab 40.9 30.4 26.8 hMN-14 2.2 1.9 0.5 Purified monocytes,monocyte-depleted PBMCs (CD14⁻CD16⁺ NK cells), or granulocytes weremixed with an equal number of Daudi cells and treated with 10 μg/mLepratuzumab or hMN-14 (anti-CEA mAb as control) for 1 h.

Example 10 Ex Vivo Trogocytosis with SLE Patient PBMCs

PBMCs were isolated from blood specimens of systemic lupus erythematosus(SLE, lupus) patients, who had yet to receive any therapy for theirdisease (naïve), and treated ex vivo with epratuzumab, using the samemethod that was applied to PBMCs from healthy donors. PBMCs of naive SLEpatients responded similarly to healthy PBMCs (as in Example 1), whereCD22, CD19, CD21 and CD79b on the surface of B cells were reduced to11±4, 53±8, 45±4 and 75±1% control, respectively (FIG. 27). Also similarto the results from normal donor PBMCs, CD27⁻ naive B cells were moreresponsive than CD27⁺ memory B cells (FIG. 28), and, a F(ab′)₂ fragmentof epratuzumab did not induce the reduction of CD19, CD21 or CD79b (FIG.29). PBMCs isolated from blood specimens of SLE patients who currentlywere on epratuzumab immunotherapy had minimal response to the ex vivotreatment with epratuzumab (not shown), presumably due to low levels ofCD22 on their B cells, resulting from therapy.

Example 11 Surface Levels of CD19, CD21, CD22 and CD79b on SLE Patient BCells on Epratuzumab Immunotherapy

The relative levels of CD22, CD19, CD21 and CD79b on B cells from fiveSLE patients who were receiving epratuzumab immunotherapy, were comparedthe results obtained from four naive lupus patients and two receivingBENLYSTA®, using identical conditions (Table 9). Only one of theepratuzumab group (S7) had a markedly reduced B cell count; however,this patient was also taking prednisone and methotrexate. Each of thefour patients on epratuzumab without methotrexate had B cell counts inthe same range as the naive patients. Both BENLYSTA® patients had low Bcell counts. As expected, CD22 was significantly (P<0.0001) lower (>80%)on the B cells of epratuzumab-treated patients (FIG. 30A). Notably,CD19, CD21 and CD79b were each significantly (P<0.02) lower for theepratuzumab group (FIG. 30B-D). We also compared the results for theepratuzumab patient specimens with those of two patients who werereceiving immunotherapy with BENLYSTA®. Although the sample size issmall, both CD19 and CD22 levels were significantly (P<0.05) lower onthe B cells of patients on epratuzumab compared to BENLYSTA®. The levelof CD21 was similarly low for the epratuzumab and BENLYSTA® patient Bcells. Because anti-CD79b-PE (instead of APC) was used to measure CD79bon B cells from BENLYSTA® patients, we could only compare these resultswith one epratuzumab patient specimen, which was measured similarly. TheCD79-PE MFI was greater for each of the BENLYSTA® specimens (MFI=425 and470) compared to that of the epratuzumab sample (MFI=186).

TABLE 9 Comparison of B cells from lupus patients % B cell in lymph-CD19 CD21 CD22 CD79b Patient Treatment gate (PE-Cy7) (FITC) (FITC) (APC)S7 E, P, M 0.5 99  9 16 186^(PE) S8 P, I 5.0 145 nd 84 nd S9 B 0.5 21821 48 470^(PE) S10 B 0.9 204 20 133 425^(PE) S11 None 18.0 195 51 106608 S12 None 13.1 160 44 114 428 S13 None 13.3 206 43 117 510 S14 None11.1 169 32 146 604 S16 E, P 8.9 128 24 27 452 S17 E, P 4.5 93 16 25 340S18 E, P 17.6 159 32 18 413 S19 E, P 20.3 155 19 38 349 E, epratuzumab;P, prednisone; M, methotrexate; I, Imuran; B, BENLYSTA ®; PE, usedinstead of APC; nd, not determined

The present studies disclose previously unknown, and potentiallyimportant, mechanisms of action of epratuzumab in normal and lupus Bcells, as well as B-cell lymphomas, which may be more pertinent to thetherapeutic effects of epratuzumab in autoimmune patients. The prominentloss of CD19, CD21, CD20, and CD79b induced by epratuzumab is not onlyFc-dependent, but also requires further engagement with FcγR-expressingeffector cells present in PBMCs. The findings of reduced levels of CD19are of particular relevance for the efficacy of epratuzumab inautoimmune diseases, because elevated CD19 has been correlated withsusceptibility to SLE in animal models as well as in patients, and lossof CD19 would attenuate activation of B cells by raising the BCRsignaling threshold. Based on these findings, the activity ofepratuzumab on B cells is two-fold, one via binding to CD22, which alsooccurs with F(ab′)₂, and the other via engagement of FcγR-bearingeffector cells. Whereas the former leads to internalization of CD22, aswell as its phosphorylation with concurrent relocation to lipid rafts(resulting in the activation of tyrosine phosphatase to inhibit theactivity of Syk and PLCr2), the latter results in the trogocytosis(shaving) of CD19, among others.

We propose that the consequences of losing CD19 from B cells are asfollows. BCR activation upon encountering membrane-bound antigeninvolves the initial spreading and the subsequent formation ofmicroclusters. Because CD19 is critical for mediating B-cell spreading,CD19-deficient B cells are unable to gather sufficient antigen totrigger B-cell activation. In addition, loss of CD19 on B cells mayseverely affect the ability of B cells to become activated in responseto T cell-dependent antigens. Thus, the epratuzumab-mediated loss ofCD19 (and possibly other BCR markers and cell-adhesion molecules) ontarget B cells may incapacitate such B cells and render themunresponsive to activation by T cell-dependent antigen. In summary,epratuzumab inactivates B cells via the loss of CD19, other BCRconstituents, and cell-adhesion molecules that are involved insustaining B-cell survival, leading to therapeutic control inB-cell-mediated autoimmune diseases. Although targeting B cells witheither epratuzumab to CD22 or rituximab to CD20 appears to share acommon effect of reducing CD19 by trogocytosis, we are currentlyinvestigating whether rituximab has a scope of trogocytosis as broad asepratuzumab. The results also caution that using CD19 as a marker forquantifying B cells by flow cytometry from patients treated with agentsthat induce CD19 trogocytosis may result in an over-estimation of B-celldepletion.

It has been shown with rituximab administered to chronic lymphocyticleukemia cells that too much antibody results in removal of complexes ofrituximab-CD20 from the leukemia cells by trogocytosis to monocytes, andcan enable these malignant cells to escape the effects of the antibodyby antigenic modulation. It was then found that reducing the dose oftherapeutic antibody could limit the extent of trogocytosis and improvethe therapeutic effects (Herrera et al., 2006). Based on our presentfindings, a similar process of antigen shaving (trogocytosis) byanti-CD22 or anti-CD20 antibodies that extends beyond the respectivetargeted antigens can be implicated in the therapy with epratuzumab orrituximab (or the humanized anti-CD20 mAb, veltuzumab). This couldexplain the clinical observations that higher doses of epratuzumabadministered to SLE or lymphoma patients did not show an improvement inefficacy over the mid-range dose used, because the concentrations ofepratuzumab in serum would be in the μM range (150 μg/mL or higher) andcould mask the low-affinity FcγRs on effector cells, thus reducing thelikely events of trogocytosis.

Example 12 Administration of Epratuzumab in Systemic Lupus Erythematosus(SLE)

An open-label, single-center study of patients with moderately activeSLE (total British Isles Lupus Assessment Group (BILAG) score 6 to 12)is conducted. Patients receive dosages of epratuzumab of 100, 200, 400and 600 mg subcutaneously (SC) every week for 6 weeks. Evaluationsinclude safety, SLE activity (BILAG), blood levels of B and T cells,human anti-epratuzumab antibody (HAHA) titers, and levels of cellsurface CD19, CD20, CD21, CD22 and CD79b on B cells. It is determinedthat a dosage of 400-600 mg per SC injection results in optimaldepletion of B cell CD19, while producing less than 50% depletion ofnormal B cells. Subsequently, a subcutaneous dose of 400 mg epratuzumabis administered to a new group of patients with moderately active SLE.

Total BILAG scores decrease by at least 50% in all patients, with 92%having decreases continuing to at least 18 weeks. Almost all patients(93%) experience improvement in at least one BILAG B- or C-level diseaseactivity at 6, 10 and 18 weeks. Additionally, 3 patients with multipleBILAG B involvement at baseline have completely resolved all B-leveldisease activities by 18 weeks. Epratuzumab is well tolerated, with noevidence of immunogenicity or significant changes in T cells,immunoglobulins or autoantibody levels. B-cell levels decrease by anaverage of 35% at 18 weeks and remain depressed for 6 monthspost-treatment.

Example 13 Prediction of Epratuzumab Response in Systemic LupusErythematosus (SLE)

Another open-label, single-center study of patients with moderatelyactive SLE is conducted. Patients receive a single dose of 400 mgepratuzumab subcutaneously. Blood levels of B and T-cells and levels ofcell surface CD19, CD20, CD21, CD22 and CD79b on B cells are determined.

Patients are divided into two groups, based on whether they show adecrease in B-cell CD19 levels above (responders) or below(non-responders) the median response for the group. It is observed thatdecreased B-cell CD19 levels are correlated with decreases in B-cellCD20, CD21, CD22 and CD79b. Subsequent s.c. administration of 400 mg ofepratuzumab occurs every week for 8 weeks and SLE activity (BILAG) ismonitored.

The group of responders shows a substantial improvement in BILAG scorescompared with the group of non-responders. Three of ten patients in theresponders group have completely resolved all BILAG B-level diseaseactivities by 18 weeks, compared with zero of ten patients in thenon-responders group. In addition, a significant improvement in totalBILAG scores is observed in the responders group compared to thenon-responders. It is concluded that trogocytosis (antigen-shaving) ofCD19 and other BCR antigens is predictive of therapeutic response totherapy with anti-CD22 antibody in SLE.

Example 14 Administration of Epratuzumab in Hairy Cell Leukemia

Patients with previously untreated or relapsed hairy cell leukemiareceive 4 doses of 80, 160, 320 or 640 mg epratuzumab injected s.c.every week or every two weeks. Occasional mild to moderate transientinjection reactions are seen with the s.c. injection and no other safetyissues are observed. The s.c. epratuzumab exhibits a slow releasepattern over several days. Transient B-cell depletion is observed at alldosage levels of epratuzumab. Depletion of B cell surface levels ofCD19, CD20, CD21, CD22 and CD79b is observed at a moderate level with320 mg and at a much higher level at 640 mg epratuzumab.

Objective responses are observed at all dose levels of s.c. epratuzumab,but with particularly high responses of 30% (mostly partial responses)at the dose of 320 mg. All serum samples evaluated for humananti-epratuzumab antibody (HAHA) are negative. Six months aftertreatment, optimal outcome is observed in the group treated with 320 mgepratuzumab, with decreased response at either higher or lower dosages.It is concluded that under these conditions, 320 mg epratuzumab is theoptimum dosage that was used. Monitoring response of BCR levels totherapeutic antibody provides an effective surrogate marker fordetermining antibody efficacy and is predictive of disease prognosis inresponse to therapy.

Example 15 Trogocytosis of BCR-Response Modulating Proteins Induced bythe RFB4 Anti-CD22 Antibody

Trogocytosis of BCR-regulating proteins, including CD19, CD21, CD22 andCD79b is assayed as described in Example 1 in response to exposure tothe anti-CD22 antibody RFB4, which binds to a different epitope of CD22than epratuzumab. Control antibody (hMN-14) is used as described inExample 1. Exposure to RFB4 antibody induces trogocytosis ofBCR-regulating proteins, similar to that induced by epratuzumab asdisclosed in Example 1. CD27⁻ naive B cells are more responsive to RFB4compared to CD27⁺ memory B cells. The effect is essentially completewithin a few hours. The reductions in surface CD19 and CD21 are notsignificantly different following 2-h or overnight treatment.

Example 16 Mechanism of Cytotoxicity Induced on Malignant B Cells byAnti-CD22 Antibody (Epratuzumab)

Summary

Epratuzumab has shown activity in patients with non-Hodgkin lymphoma,systemic lupus erythematosus, and Sjögren's syndrome, but the mechanismby which it depletes B cells in vivo has previously been unknown. Invitro, epratuzumab is cytotoxic to CD22-expressing human Burkittlymphoma lines only when immobilized onto plastic plates or combinedwith a secondary antibody plus anti-IgM.

We used a Daudi lymphoma subclone selected for high expression ofmembrane IgM (mIgM) to investigate the cytotoxic mechanism ofimmobilized epratuzumab, and showed that it induced similarintracellular changes as observed upon crosslinking mIgM with anti-IgM.Specifically, we identified phosphorylation of CD22, CD79a and CD79b,and their translocation to lipid rafts, as essential for cell killing.Other findings include the co-localization of CD22 with mIgM, formingcaps before internalization; induction of caspase-dependent apoptosis(25-60%); and a pronounced increase of pLyn, pERKs and pJNKs with aconcurrent decrease of constitutively-active p38. The apoptosis waspreventable by JNK or caspase inhibitors, and involved mitochondrialmembrane depolarization, generation of reactive oxygen species,upregulation of pro-apoptotic Bax, and downregulation of anti-apoptoticBcl-xl, Mcl-1 and Bcl-2. These findings indicated, for the first time,that epratuzumab and anti-IgM behave similarly in perturbing multipleBCR-mediated signals in malignant B cells.

Introduction

Epratuzumab (hLL2), a humanized anti-CD22 monoclonal antibody, iscurrently under clinical investigation for the treatment of non-Hodgkinlymphoma (NHL) and systemic lupus erythematosus (SLE). CD22, alsoreferred to as sialic acid-binding Ig-like lectin-2 (Siglec-2) orB-lymphocyte adhesion molecule (BL-CAM), is a transmembrane type-Iglycoprotein of 140 kDa, widely and differentially expressed on B cells(Kelm et al., 1994, Curr Biol 4:965-972; Law et al., 1995, J Immunol155:3368-76; Wilson et al., 1991, J Exp Med 173:137-46). Structurally,the extracellular portion of CD22 comprises 7 Ig-like domains, of whichthe two N-terminal domains are involved in ligand binding, while thecytoplasmic tail contains 6 conserved tyrosine residues localized withinthe immunoreceptor tyrosine-based inhibition motifs (ITIM) andimmunoreceptor tyrosine-based activation motifs (ITAM) (Wilson et al.,1991, J Exp Med 173:137-46; Schulte et al., 1992, Science 258:1001-4;Torres et al., 1992, J Immunol 149:2641-49). Functionally, CD22recognizes α2,6-linked sialic acids on glycoproteins in both cis (on thesame cell) and trans (on different cells) locations, and modulates Bcells via interaction with CD79a and CD79b, the signaling components ofthe B-cell receptor (BCR) complex (Leprince et al., 1993, Proc Natl AcadSci USA 90:3236-40; Peaker et al., 1993, Eur J Immunol 23:1358-63).Crosslinking BCR with cognate antigens or appropriate antibodies againstmembrane immunoglobulin (mIg) on the cell surface induces translocationof the aggregated BCR complex to lipid rafts, where CD79a, CD79b andCD22, among others, are phosphorylated by Lyn (Marshall et al., 2000,Immunol Rev 176:30-46; Niiro et al., 2002, Nat Rev Immunol 2:945-56;Smith et al., 1998, J Exp Med 187:807-11), which in turn triggersvarious downstream signaling pathways, culminating in proliferation,survival, or death (Peaker et al., 1993, Eur J Immunol 23:1358-63; Niiroet al., 2002, Nat Rev Immunol 2:945-56; Pierce & Liu, 2010, Nat RevImmunol 10:767-77). Importantly, phosphorylated CD22, depending onenvironmental cues, can either positively or negatively affectBCR-mediated signaling pathways (Niiro et al., 2002, Nat Rev Immunol2:945-56; Pierce & Liu, 2010, Nat Rev Immunol 10:767-77; Nitschke, 2005,Curr Opin Immunol 17:290-97; Otipoby et al., 2001, J Biol Chem276:44315-22). Understanding the role of CD22 in B-cell malignancies, aswell as B-cell-implicated autoimmune diseases, is of considerableinterest.

As a single agent, epratuzumab is well-tolerated and depletescirculating B cells in patients with NHL, SLE, and Sjögren's syndrome by35 to 50% (Goldenberg, 2006, Expert Rev Anticancer Ther 6:1341-53;Leonard & Goldenberg, 2007, Oncogene 26:3704-13; Leonard et al., 2003, JClin Oncol 21:3051-59; Leonard et al., 2004, Clin Cancer Res 10:5327-34;Dorner et al., 2006, Arthritis Res Ther 8:R74). It has modestantibody-dependent cellular cytotoxicity (ADCC) but nocomplement-dependent cytotoxicity in vitro (Carnahan et al., 2007, MolImmunol 44:1331-41). In vivo, it targets CD27⁻ naive and transitional Bcells, and decreases surface CD22 expression (Jacobi et al., 2008, AnnRheum Dis 67:450-57). Epratuzumab downregulates the surface expressionof certain adhesion molecules (CD62L and β7 integrin), and increases theexpression of β1 integrin on CD27⁻ B cells, resulting in migration of Bcells towards the chemokine, CXCL12 (Daridon et al., 2010, Arthritis ResTher 12:R204). Soluble epratuzumab does not have cytotoxic or cytostaticeffects in vitro or in xenografts of human lymphoma in vivo (Carnahan etal., 2007, Mol Immunol 44:1331-41; Carnahan et al., 2003, Clin CancerRes 9:3928S-90S; Stein et al., 1993, Cancer Immunol Immunother37:293-98). However, when immobilized to plastic plates or added incombination with suboptimal amounts of anti-IgM along with acrosslinking secondary antibody, it induces growth-inhibition in NHLcell lines, such as Ramos and Daudi (D1-1), a subclone of Daudi selectedfor a high expression of BCR (Qu et al., 2008, Blood 111:2211-19). Wehave reported previously that soluble epratuzumab phosphorylates andtranslocates CD22 to lipid rafts upon engagement (Qu et al., 2008, Blood111:2211-19), but the exact mechanism by which epratuzumab kills normaland malignant B cells in patients, and inhibits the growth of lymphomalines in vitro upon immobilization, remains elusive.

In this study, we evaluated key signaling pathways and moleculesaffected by immobilized epratuzumab. We showed in D1-1 cells thatepratuzumab by either non-covalent adsorption on microtiter plates orconjugated covalently to polystyrene beads induces phosphorylation ofCD22, CD79a and CD79b, and their translocation to lipid rafts, which areinstrumental for cell death via caspase-dependent apoptosis. Additionalexperiments showed that immobilization of epratuzumab also inducessubstantial apoptosis (25 to 60%) in Ramos lymphomas. A pronouncedphosphorylation of ERK and JNK MAP kinases, accompanied by a decrease inphosphorylated p38 MAP kinase, also was observed. Selective experimentsinterrogating intracellular events identified changes in mitochondrialmembrane potential, generation of reactive oxygen species (ROS),involvement of caspases, and modulation of pro- and anti-apoptoticproteins, in the mechanisms of immobilized epratuzumab.

Materials and Methods

Cell Lines, Antibodies, and Reagents— The Burkitt lymphoma cell lines,Daudi and Ramos, were obtained from ATCC (Manassas, Va.). D1-1, asubclone of Daudi selected for a higher expression of the BCR, wasdeveloped in-house (Qu et al. 2008, Blood 111:2211-19). Phospho-specificand other antibodies were obtained from CELL SIGNALING TECHNOLOGY®(Danvers, Mass.) and SANTA CRUZ BIOTECHNOLOGY® (Santa Cruz, Calif.).Anti-tyrosine antibody 4G10 was bought from Millipore (Billerica,Mass.), anti-IgM antibody, secondary goat anti-human Fc specific andrhodamine conjugated F(ab′)₂ fragment goat anti-human IgG, F(ab′)₂fragment specific were obtained from Jackson ImmunoResearch (West Grove,Pa.). Cell culture media, supplements, annexin V ALEXA FLUOR® 488conjugate, TMRE, and CM-H₂DCF-DA were supplied by INVITROGEN™ (GrandIsland, N.Y.). One Solution Cell Proliferation assay reagent wasobtained from Promega (Madison, Wis.). PHOSPHOSAFE™ and RIPA bufferswere procured from EMD chemicals (Billerica, Mass.). For epratuzumabimmobilization, non-tissue-culture flat-bottom polystyrene plates wereobtained from BD Biosciences (San Jose, Calif.), and CP-30-10carboxyl-coated polystyrene beads were bought from Spherotech (LakeForest, Ill.). All other chemicals were obtained from SIGMA-ALDRICH®(St. Louis, Mo.).

Immobilization of Epratuzumab— Epratuzumab (10 μg/mL or as indicated) incarbonate/bicarbonate buffer (50 mM; pH 9.6) was immobilized onnon-tissue-culture flat-bottom plates by incubating the plate at 4° C.overnight. Next day, plates were washed 2× with RPMI-1640 medium.Besides immobilizing epratuzumab onto plates, 100 μg was alsoimmobilized to Protein A beads (100 μL). Supernatants were analyzed forthe amounts of epratuzumab bound to the beads. Epratuzumab-bound beadswere washed 3× with PBS and reconstituted in 100 μL of the RPMI-1640medium. For flow cytometry, epratuzumab also was conjugated to CP-30-10carboxyl-coated polystyrene beads using the manufacturer's protocol.Briefly, 50 μg of epratuzumab was conjugated to 200 μL of polystyrenebeads in 1 mL of MES buffer containing 20 mg of EDC for 30 min. Beadswere washed 3× with PBS and reconstituted in 0.05M IVIES buffercontaining 0.05% BSA.

Cell Culture and Cytotoxicity Assay— Cell lines were cultured inRPMI-1640 medium supplemented with 10% heat-inactivated fetal bovineserum (FBS), 2 mM L-glutamine, 200 U/mL penicillin, and 100 μg/mLstreptomycin in a humidified incubator at 37° C. with 5% CO₂. Toevaluate the functional activity of epratuzumab or epratuzumab F(ab′)₂,different amounts (5, 10 and 20 μg/mL) were immobilized in 48-wellplates. Plates were washed and D1-1 or Ramos cells were seeded (1×10⁴cells per well) and incubated for 4 days. The number of viable cells wasthen determined using the MTS assay per the manufacturer's protocol,plotted as percent of the untreated. Activity of soluble epratuzumab orepratuzumab F(ab′)₂ was also evaluated.

Annexin V Binding Assay— Cells in 6-well plates (2×10⁵ cells per well)were either treated with epratuzumab immobilized to polystyrene beads orimmobilized to plates for 24 or 48 h, washed, resuspended in 100 μl ofannexin-binding buffer, and stained with 5 μl of Annexin V-ALEXA FLUOR®488 conjugate for 20 min. Cells were then stained with 1 μg/mL propidiumiodide (PI) in 400 μl of annexin-binding buffer, and analyzed by flowcytometry (FACSCALIBUR™). When required, cells were pretreated with theindicated inhibitors for 2 h before adding the test article.

Immunoblot Analysis— D1-1 and Ramos cells (2×10⁷ cells) were added toplates immobilized with epratuzumab (10 μg/mL) for varying time pointsas indicated. Cells were washed with PBS, lysed in ice-cold PHOSPHOSAFE™buffer, and the lysates clarified by centrifugation at 13,000×g. Proteinsamples (25 μg/lane) were resolved by SDS-PAGE on 4-20% gradienttris-glycine gels followed by transfer onto nitrocellulose membranes.

Isolation of Lipid Rafts— D1-1 cells (3×10⁷) were treated with theindicated antibodies or added to plates coated with epratuzumab (10μg/mL) for 2 h. After treatment, cells were lysed in 2 mL of buffercontaining CHAPS/low-salt (20 mM NaCl and 40% sucrose), and lysates werefractionated in a sucrose gradient and lipid rafts were prepared asdescribed earlier (Qu et al., 2008, Blood 111:2211-19).

Co-Immunoprecipitation Analysis— Six-well plates were coated with therequired antibodies (10 μg/mL) in carbonate/bicarbonate buffer for 24 h.Plates were washed with RPMI-1640 medium containing 5% FBS, and D1-1cells were added to the wells (5×10⁶ cell/well) for 2 h. Followingincubation, cells were lysed in ice-cold RIPA buffer, andco-immunuprecipitation was performed using phospho-tyrosine antibody(4G10; 1:200 dilution), as described earlier (Gupta et al., 2006, CancerRes 66:8182-91). 20 μl of the samples were separated by SDS-PAGE andtransferred onto a nitro-cellulose membrane, followed by probing withthe indicated antibodies.

Mitochondrial Membrane Potential (Δψ_(m)) Reactive Oxygen SpeciesAssays— D1-1 cells (2×10⁵ cells per well) were added to the 6-wellplates coated with epratuzumab (10 μg/mL) for 48 h. Cells were washedand stained for 30 min in the dark at 37° C., either with TMRE (50 nM)for Δψ_(m) analysis or CM-H₂DCF-DA (1 μM) for ROS analysis. Samples werewashed 3× with PBS and analyzed for changes in fluorescence using flowcytometry.

Immunofluorescence Analysis— To analyze the co-localization of CD22 andIgM receptors, D1-1 cells were treated with epratuzumab (7.5 μg/mL) oranti-IgM conjugated to ALEXA FLUOR® 488 (1 μg/mL) alone and incombination with a secondary crosslinking goat anti-human antibody for 5min at 37° C. Cells were washed with PBS to remove the antibodies andincubated at room temperature for 30 min, followed by fixation with 4%paraformaldehyde, and staining with rhodamine-conjugated Fc-specificgoat anti-human IgG for 20 min. Cells were washed with PBS andvisualized by fluorescence microscopy. To evaluate the translocation ofCD22 in lipid rafts, D1-1 cells were incubated with ProteinA-immobilized epratuzumab for 4 h, fixed, and permeabilized with 0.1%Triton X-100 in PBS. CD22 and IgM receptors were evaluated byepratuzumab-dylight 550 and anti-IgM-ALEXA FLUOR® 488, respectively.Images were overlaid using Photoshop software.

Cell Cycle Analysis— Cells were seeded in 6-well plates (2×10⁵ cells perwell) and treated with epratuzumab conjugated to polystyrene beads orthe indicated antibodies for 72 h. Following incubation, cell cycleanalysis was performed by flow cytometry as described (Gupta et al.,2010, Blood 116:3258-67).

Results

Immobilization of Epratuzumab Induces Growth-Inhibition and Apoptosis—The ability to induce growth-inhibition was evaluated by immobilizingepratuzumab to non-tissue-culture coated flat-bottom plates. Varyingamounts of epratuzumab were immobilized. In the cell viability assay, 5μg/mL of immobilized epratuzumab induced significant growth-inhibitionin D1-1 cells (FIG. 31A). About 60% growth-inhibition was observed atthis concentration, and little change was found at higher concentrationsof 10 and 20 μg/mL, indicating saturation (FIG. 31A). Similargrowth-inhibition of the Burkitt lymphoma line, Ramos, was observed,although it was slightly less than with D1-1. In Ramos cells, 10 μg/mLepratuzumab induced about 45% growth-inhibition (FIG. 31A). Thisdifference in sensitivity could be due to the levels of CD22 andoverexpression of BCR components in D1-1. Immobilized nonspecific hMN-14antibody did not induce growth-inhibition in either cell line (FIG.31A). Soluble epratuzumab in the media, even at the highestconcentration (20 μg/mL), did not induce growth-inhibition in eithercell line, indicating the requirement of immobilization (FIG. 31B).

We next evaluated the role of apoptosis in the effect of epratuzumab.Carboxyl-coated polystyrene beads were used to immobilize epratuzumab.As shown in FIG. 31C, 5 and 20 μL of epratuzumab-coated beads inducedapoptosis in both D1-1 and Ramos at 24 h. In D1-1, 5 μL ofepratuzumab-coated beads induced about 75% apoptosis, while similaramounts of uncoated beads displayed annexin V staining, comparable tountreated cells (FIG. 31C). Significant apoptosis was also observed inRamos cells by epratuzumab-coated beads (FIG. 31C). Likewise, ProteinA-immobilized epratuzumab induced apoptosis and growth-inhibition inboth D1-1 and Ramos cells (data not shown). These results demonstratethe requirement of epratuzumab immobilization onto plastic or to beadsfor inducing growth-inhibition and apoptosis in the target malignantcells. Similar to epratuzumab, the immobilized F(ab′)₂ fragments ofepratuzumab also induced apoptosis and growth inhibition in D1-1 cells(FIG. 31D). These results negate the role of Fc effector functions andconfirm the role of signaling events in the target cells for observedgrowth inhibition though immobilization.

Immobilized Epratuzumab Induces Phosphorylation of CD22, CD79a andCD79b— To understand the mechanism by which immobilized epratuzumabinhibits growth in these lymphoma lines, we evaluated thephosphorylation profiles of the BCR components, CD79a and CD79b. CD79aand CD79b form hetrodimers and are noncovalently-bound membraneimmunoglubulins that regulate BCR-mediated signaling by ITAM motifs intheir cytoplasmic tails. Cells were subjected to immobilized epratuzumaband other antibodies for 2 h, and co-immunoprecipitation experimentswere performed using the phospho-tyrosine antibody, 4G10. As shown inFIG. 32A (top panel), anti-IgM (10 μg/mL) antibody inducedphosphorylation of CD22, CD79a and CD79b molecules, while solubleepratuzumab induced phosphorylation of CD22, but not CD79a and CD79b.Immobilization of anti-IgM and epratuzumab induced phosphorylation ofCD22 as well as CD79a and CD79b (FIG. 32A; bottom panel). Ligation ofCD22 on D1-1 by immobilized epratuzumab was similar to ligation of BCRby anti-IgM (above a threshold concentration, i.e., 10 μg/mL), in thatboth resulted in the phosphorylation of CD22, CD79a and CD79b. Similarphosphorylation of CD22, CD79a and CD79b was observed with solubleepratuzumab combined with suboptimal amounts of anti-IgM (1 μg/mL) and asecondary crosslinking goat anti-human IgG, while anti-IgM (1 μg/mL)alone did not induce phosphorylation of any of these molecules (FIG.32B). Soluble epratuzumab in combination with anti-IgM and a secondarycrosslinking antibody has been observed previously to inducegrowth-inhibition in lymphoma lines. These results with respect todifferences in the phosphorylation profiles of CD79a and CD79b bysoluble and immobilized epratuzumab clearly implicate components of BCRin the growth-inhibition due to immobilized epratuzumab or thecombination of epratuzumab and anti-IgM antibody.

Immobilized Epratuzumab Translocates CD22 and CD79 to Lipid Rafts— Theobservation that immobilized epratuzumab induces phosphorylation of BCRcomponents, CD79a and CD79b, prompted us to investigate the membranedistribution of CD22, CD79a and CD79b in lipid rafts, using sucrosedensity gradient ultracentrifugation. Anti-IgM (10 μg/mL) treatmentresulted in the distribution of CD22, CD79a and Cd79b into lipid rafts(FIG. 32C). Soluble epratuzumab, which is known to inducephosphorylation of CD22 and migration of CD22 into lipid rafts (Qu etal., 2008, Blood 111:2211-19), did not induce redistribution of CD79aand CD79b into lipid rafts (FIG. 32C). However, soluble epratuzumabtogether with suboptimal amounts of anti-IgM (1 μg/mL) and a secondarycrosslinker resulted in the migration of CD22, CD79a and CD79b intolipid rafts. Since soluble epratuzumab together with anti-IgM (1 μg/mL)and a crosslinker induced growth-inhibition in these malignant cells,the presence of phosphorylated CD22, CD79a and CD79b in lipid raftsseems to be critical for the effects of epratuzumab. Immobilizedepratuzumab also induced migration of these components into lipid rafts,although the signals were not as strong as they were for other samples;this could be due to loss of some treated cells because of adherence tothe epratuzumab-coated plates (FIG. 32C).

We also examined the distribution of CD22 and BCR components byimmunofluorescence. Soluble epratuzumab binds to CD22 and internalizesrapidly into the cells (Carnahan et al., 2003, Clin Cancer Res9:3982S-90S). To study the distribution of CD22 and IgM receptors, wetreated the cells with different antibodies alone or in combination for5 min at 37° C. Cells were fixed after 30 min. Imunofluorescenceanalysis revealed the binding of soluble epratuzumab and anti-IgM tocell-surface CD22 and IgM receptors, respectively, when the twoantibodies were evaluated separately (FIG. 32D). However, when solubleepratuzumab combined with suboptimal amounts of anti-IgM (1 μg/mL) wereadded, they formed caps and co-localized in about 70% of cells (FIG.32D). Similar co-localization of CD22 and IgM receptors was observedwhen cells were treated with Protein A-bound epratuzumab (FIG. 32E).These observations indicate the co-localization and requirement of bothIgM and CD22 receptors, either when soluble epratuzumab is used togetherwith suboptimal amounts of anti-IgM or when epratuzumab is immobilized.

Requirement of Lyn for Growth-Inhibition by Immobilized Epratuzumab— Lynplays a critical role in regulating BCR activity by phosphorylatingtyrosine residues in the ITAM domain of CD79a, CD79b, and ITIM domain inCD22, followed by recruitment of SHP-1 to CD22 (Schulte et al., 1992,Science 258:1001-4; Nitschke, 2005, Curr Opin Immunol 17:290-97;Chaouchi et al., 1995, J Immunol 154:3096-104; Doody et al., 1995,Science 269:242-44; Nitschke 2009, Immunol Rev 230:128-43). Tounderstand this growth-inhibition, we evaluated the phosphorylationprofiles of Lyn as a function of time. D1-1 cells were added toepratuzumab-coated plates for different times up to 4 h. Cells werelysed in RIPA buffer and phospho-tryosine residues wereimmunoprecipitated using monoclonal antibody 4G10. Immobilizedepratuzumab induced rapid phosphorylation of tyrosine residues thatcontinued for 4 h (not shown). Probing the same membranes with differentantibodies depicted rapid and sustained phosphorylation of Lyn and Sykmolecules (not shown). In a separate experiment, we repeated thesestudies until 24 h, and observed that immobilized epratuzumab inducesthe phosphorylation of Lyn and PLCy2 (not shown). Although we observedphosphorylation of Syk by co-immunoprecipitation, we did not observe asimilar time-dependent phosphorylation of Syk by using anti-phospho Sykantibodies (not shown).

To further elucidate the role of Lyn in this growth-inhibition byimmobilized epratuzumab, we evaluated the binding of SHP-1 to thetyrosine residues. Cells were treated with various antibody combinationsand a co-immunoprecipitation experiment was performed using antibody4G10. Membranes were probed with SHP-1 antibody and the results indicatebinding of SHP-1 to tyrosine residues in the samples treated withimmobilized epratuzumab (not shown). Similar binding of SHP-1 wasobserved in samples treated with epratuzumab and suboptimal amounts ofanti-IgM in presence of a secondary crosslinking antibody (not shown).In contrast, no significant binding was observed in samples treated withsoluble epratuzumab or suboptimal amounts of anti-IgM alone (not shown).These results establish the requirement of phosphorylation of Lyn andrecruitment of SHP-1 to CD22 to negatively regulate BCR signalingresulting in growth-inhibition.

Modulation of MAP Kinases— Mitogen-activated protein (MAP) kinases are agroup of serine threonine protein kinases that respond to a variety ofenvironmental cues, such as growth factors, cellular stress (e.g., UV,osmotic shock, DNA damage) and others, by either inducing survival andcell growth, or apoptosis. Previously, we observed that the anti-HLA-DRmAb, IMMU-114, induced growth-inhibition by hyperactivation of the ERKand JNK group of MAP kinases, while p38 was not affected (Stein et al.,2010, Blood 115:5180-90). To further elucidate the mechanism ofgrowth-inhibition by immobilized epratuzumab, we studied the effects onall three MAP kinases. Immobilized epratuzumab induced modest activationand phosphorylation of the ERK and JNK group of MAP kinases (not shown).This activation was rapid, and could be detected within 30 min andsustained over a period of 24 h. In contrast, p38, the third group ofMAP kinases, was inhibited and the phoshorylation of p38 wasdownregulated by immobilized epratuzumab within 30 min of treatment ofthe target cells (not shown).

We further studied the role of stress in the growth-inhibition byimmobilized epratuzumab in the presence of an inhibitor ofstress-activated JNK MAP kinase, SP600125. Two doses (2.5 and 5 nM) ofthe inhibitor were evaluated and at both doses, apoptosis was inhibitedsignificantly in D1-1 cells (FIG. 3). Thus, this differentialactivation/inhibition of MAP kinases attests to the fact thatimmobilized epratuzumab affects target cells by invoking multiplesignaling pathways.

Immobilized Epratuzumab Induces Production of ROS and Changes inMitochondrial Membrane Potential— Induction of stress in cells resultsin the generation of free oxygen radicals in mitochondria. ROS arechemically-reactive oxygen molecules that induce mitochondrial membranedepolarization, activating pro-apoptotic proteins such as Bax, andresulting in programmed cell death in the target cells. To furtherinvestigate the role of stress in this growth-inhibition by immobilizedepratuzumab, we studied the generation of ROS and changes inmitochondrial membrane potential in the affected cells. Treatment withimmobilized epratuzumab resulted in about 24% cells having enhanced ROSproduction compared to about 10% in D1-1 cells treated with solubleepratuzumab or untreated (not shown).

Immobilized epratuzumab induced mitochondrial membrane depolarization inabout 45% of D1-1 cells, compared to about 20% of cells treated withimmobilized nonspecific hMN-14 antibody or untreated (not shown).Similar results for ROS and changes in mitochondrial membrane potentialwere observed in Ramos (data not shown).

Immobilized Epratuzumab Induces Caspase-Mediated Apoptosis— We nextevaluated the effect of immobilized epratuzumab on pro-/anti-apoptoticproteins and caspases in D1-1 and Ramos cells subjected to immobilizedepratuzumab for 24, 48 and 72 h. Cell lysates were evaluated for theexpression profiles of anti-apoptotic proteins, Bcl-2, Bcl-xL and Mcl-1,and pro-apoptotic protein, Bax. In both cell lines, immobilizedepratuzumab downregulated anti-apoptotic proteins, Bcl-xL and Mcl-1, andincreased the expression levels of pro-apototic, Bax (not shown). Bcl-2was downregulated in D1-1, and very low levels were detected in Ramos.The observed apoptosis by immobilized epratuzumab in both D1-1 and Ramoswas caspase-dependent, as observed by the cleavage of caspase 3, caspase9 and PARP molecules, which are known to induce apoptosis in the targetcells (not shown). The observed apoptosis was abrogated by thepan-caspase inhibitor, z-vad-fmk (10 μM) in D1-1, confirming therequirement of caspases in the apoptosis induced by immobilizedepratuzumab (FIG. 4).

Deregulation of the Cell Cycle— Immobilized epratuzumab was observed toarrest D1-1 cells in G1 phase of the cell cycle (not shown), whilesoluble epratuzumab had no effect. Epratuzumab conjugated to beadsresulted in about 10% more cells in the G1 phase. A similar increase inthe levels of cells was observed in samples treated with anti-IgM orepratuzumab combined with suboptimal amounts of anti-igM. Thisderegulation of the cell cycle was associated with changes in the levelsof CDK inhibitors, such as p21, p27, and p57 and expression levels ofcyclin D1, Rb and phosphorylation of Rb (not shown).

Calcium Release Assay— We did not observe any release of calcium byimmobilized epratuzumab. Also, we did not find an inhibitory effect ofepratuzumab or immobilized epratuzumab on the anti-IgM-mediated releaseof calcium, even after preincubating the cells for 18 h (not shown).

Discussion

In the present study, we confirmed that ligation of mIgM by a sufficientamount of anti-IgM (10 μg/mL) induces the phosphorylation of CD22, CD79aand CD79b, and the localization of all three phosphorylated proteins inthe lipid rafts, leading to cell death in the Burkitt D1-1 line. Wefurther show that ligation of CD22 with immobilized epratuzumab inducesa similar change in CD22, CD79a and CD79b, including phosphorylation,translocation into lipid rafts, and subsequent cell death. Thus, itappears that for a CD22-binding agent to kill Daudi cells in particular,and perhaps other CD22-expressing B-cell lymphomas, two critical eventsmust occur in concert, (i) phosphorylation of CD22, CD79a and CD79babove a threshold level, and (ii) their movement to lipid rafts. Thisnotion is supported by the finding that little or no cell death wasobserved for D1-1 with either soluble epratuzumab at 50 nM plus asecondary crosslinking antibody or with a suboptimal amount of anti-IgM(1 μg/mL). The former treatment efficiently induced phosphorylation ofCD22 and the localization of phospho-CD22 into lipid rafts, but wasunable to translocate the weakly phosphorylated CD79a and CD79b to lipidrafts, whereas the latter treatment failed to phosphorylate CD22, CD79aand CD79b at all. On the other hand, combining these two treatmentscould effect both phosphorylation of CD22, CD79a and CD79b, along withtheir localization into lipid rafts, and consequently, cell death, asobserved for anti-IgM at 10 μg/mL or immobilized epratuzumab.

Binding of CD22 to beads coated with B3 antibody for human CD22 wasreported to lower the threshold concentration of anti-IgM required forstimulating DNA synthesis in tonsillar B cells by two orders ofmagnitude, presumably due to sequestration of CD22 from mIgM byrestricting the lateral movement of CD22 in the plane of the cellmembrane (Doody et al., 1995, Science 269:242-44). Ourimmunofluorescence results obtained with D1-1 cells, however, showotherwise, as demonstrated by the colocalization of mIgM and CD22 into acap-like structure with both soluble epratuzumab and anti-IgM added, andan even more massive coaggregation with epratuzumab immobilized onbeads. Thus, we believe that the ability of immobilized epratuzumab topromote such a high degree of mIgM crosslinking without the need foranti-IgM constitutes a sufficient condition for cell killing and negatesthe inhibitory effect of phosphorylated CD22 in close proximity.

Knowing that binding of CD22 by soluble epratuzumab leads to promptinternalization, and engagement of CD22 with epratuzumab immobilized onplastics should not, raises the question whether internalization of CD22plays a role in the mechanism of cell killing. Also, the intracellularfate of CD22 after internalization needs to be addressed withexperiments designed to determine the kinetics of CD22 recycling, whichmay reveal that internalized CD22 is predominantly degraded, rather thanrecycled.

Taking a cue from CD20, which also interacts with BCR and affectscalcium mobilization (Walshe et al., 2008, J Biol Chem 283:16971-84) andits own degradation Kheirallah et al., 2010, Blood 115:985-94), theexpression levels of CD22 as well as BCR on the cell surface may becritical for the activity of anti-CD22 mAbs, in particular for anon-blocking anti-CD22 mAb like epratuzumab.

Intriguingly, we neither observed any transient increase inintracellular calcium by immobilized epratuzumab nor any inhibitoryeffect of immobilized epratuzumab on calcium release after stimulationwith anti-IgM (not shown). Experiments with longer incubation (16 h) ofimmobilized epratuzumab followed by stimulation with anti-IgM also didnot have any effect on resulting calcium release (not shown). Theseresults were corroborated by a recent finding that a multivalentsialylated polymer synthesized to bind only CD22, but not mIgM, failedto induce any calcium flux (Courtney et al., 2009, Proc Natl Acad SciUSA 106:2500-5), and highlight a key dissimilarity between the mechanismof anti-IgM and immobilized epratuzumab is calcium mobilization, whichmay require direct engagement of mIgM with anti-IgM. However,resemblances of anti-IgM and immobilized epratuzumab in theircharacteristic mechanism of action abound, as demonstrated by a similarprofile of signal alterations in ERKs, JNKs and p38 MAPK,caspase-dependent apoptosis, change in mitochondria membrane potential,and the generation of ROS.

In conclusion, we provide evidence for the mechanism of action by whichimmobilized epratuzumab induces cytotoxic and cytostatic effects inCD22-expressing B lymphoma lines (D1-1 and Ramos), both of which haveBCR of the IgM isotype. These findings indicate, for the first time,that immobilized epratuzumab and anti-IgM behave similarly in perturbingthe BCR-mediated signals in malignant B cells.

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated herein by reference,including any Tables and Figures, to the same extent as if eachreference had been incorporated by reference individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the invention.

What is claimed is:
 1. A method of treating a human patient with anautoimmune disease selected from the group consisting of systemic lupuserythematosus (SLE) and Sjogren's syndrome comprising: a) exposing Bcells from the human patient to a chimeric, humanized or human anti-CD22antibody in vitro in the presence of PBMCs (peripheral blood mononuclearcells) or FcγR-positive cells; b) measuring the depletion of one or moreantigens selected from the group consisting of CD19, CD20, CD21, CD22and CD79b on the surface of the B cells, wherein the antigens aredepleted by trogocytosis, to determine the sensitivity of the B cells tothe anti-CD22 antibody; and c) administering the anti-CD22 antibody tothe human patient, wherein the antibody is capable of inducingtrogocytosis of the one or more antigens from the patient's B cells. 2.The method of claim 1, wherein the patient has not previously beentreated with an anti-CD22 antibody.
 3. The method of claim 1, whereinthe anti-CD22 antibody is epratuzumab.
 4. The method of claim 1, whereinthe anti-CD22 antibody is a naked antibody.
 5. The method of claim 1,wherein the antigen is CD19.
 6. The method of claim 1, wherein theautoimmune disease is Sjogren's syndrome selected from the groupconsisting of acute idiopathic thrombocytopenic purpura, chronicidiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndrome, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitides,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjögren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis, dermatomyositis, polychondritis, bullouspemphigoid, pemphigus vulgaris, Wegener's granulomatosis, membranousnephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis, polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis and fibrosing alveolitis.
 7. The method ofclaim 1, wherein the autoimmune disease is systemic lupus erythematosus(SLE).